Atherosclerosis, 1996 Sep 27, 126(1), 95 - 104 Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in rabbit aortic neointima after selective de-endothelialization; Wang H et al.; Altered TIMP-1 synthesis in the arterial wall may be important for the balance between metalloproteinases and their inhibitors, and thus contribute to dysregulated extracellular matrix metabolism in atherosclerotic lesions . To examine this, we cloned the rabbit TIMP-1 gene from aortic neointima, developed in response to a balloon-catheter induced de-endothelialization . The apparent homology of cDNA with TIMP-1 genes from several sources suggested that it is a rabbit form of TIMP-1 . We examined the recombinant rabbit TIMP-1 expression in Escherichia coli using the pTrxFus expression system and the synthesis of the resulting soluble protein was confirmed by immunostaining with anti-TIMP-1 . The TIMP-1 concentration in normal and de-endothelialized rabbit aortas was compared using Northern blot, Western blot and mRNA in situ hybridization techniques . We observed a significant increase of TIMP-1 expression in neointimal SMCs at both nucleic acid and protein levels, suggesting a role of TIMP-1 in injury-induced atherogenesis. Biochim Biophys Acta, 1996 Sep 27, 1303(2), 92 - 102 Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli: characterisation of N-terminal mutants; Othman R et al.; A gene coding for human non-pancreatic (group II) secreted phospholipase A2 (hnpsPLA2) has been constructed by the single-step ligation of twelve synthetic oligonucleotides . The gene has been cloned into a modification of the bacterial expression vector pET 11 which allows protein over-expression as inclusion bodies and enables about 3 mg/litre of pure refolded fully active enzyme to be obtained . The protein was expressed as a 1-Ala mutant (N1A) to allow removal of the initiator methionine by the Escherichia coli amino-peptidase . This mutant had very similar properties to the wild-type enzyme . A double mutant, N1A, V3W was also constructed and expressed in high yield . This tryptophan-containing mutant showed similar properties to the wild-type and N1A mutant but had about 40% of the activity under the assay conditions used . This tryptophan was used as a reporter group for interfacial binding and its properties were compared to those of the corresponding tryptophan in PLA2 from procine pancreas . Expression of the wild-type gene sequence for hnpsPLA2 in E . coli gave the expected mutant protein still with the initiator methionine and with much reduced activity . Interfacial binding of all hnpsPLA2 mutants to anionic phospholipids was very similar when assessed by fluorescence methods . Comparisons of these mutants with the pancreatic enzyme revealed significant differences in terms of the effect of calcium on interfacial binding . The ability to express reasonably large amounts of the N1A mutant in E . coli will provide a basis for future site directed mutagenesis studies of this important human enzyme. J Mol Biol, 1996 Sep 27, 262(3), 375 - 86 A new subclass of the zinc metalloproteases superfamily revealed by the solution structure of peptide deformylase; Meinnel T et al.; Escherichia coli peptide deformylase, a member of the zinc metalloproteases family, is made up of an active core domain composed of 147 residues and of an additional and dispensable C-terminal tail of 21 residues . The three-dimensional structure of the catalytic core could be studied by NMR . 1H and 15N NMR resonances assignments were obtained by two-dimensional and three-dimensional heteronuclear spectroscopy . The structure could be calculated using a set of 1015 restraints for the 147 residues of the enzyme . The overall structure is composed of a series of antiparallel beta-strands which surround two perpendicular alpha-helices . The C-terminal helix contains the HEXXH motif, which is crucial for activity . This helical arrangement and the way the histidines bind the zinc ion clearly are structurally reminiscent of the other members of the metalloprotease family, such as thermolysin or metzincins . Nevertheless, the overall arrangement of secondary and tertiary structures of peptide deformylase and the positioning of its third zinc ligand (a cysteine) are quite different from those of the other members of the family . These discrepancies, together with several biochemical differences, lead us to propose that peptide deformylase is the first example of a new class of the zinc-metalloproteases family . Studies of the interaction of peptide deformylase with either an inhibitor of the reaction or a product of the catalysed reaction, Met-Ala-Ser, as well as comparisons with the structures of other enzymes of the family, have enabled us to delineate the area corresponding to their binding site . The structural basis of the specificity of recognition of the formyl group is discussed in the context of the protease superfamily. J Biol Chem, 1996 Sep 27, 271(39), 24138 - 43 Catalytic mechanism and DNA substrate recognition of Escherichia coli MutY protein; Lu AL et al.; Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities . In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity . To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed . MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates . The catalytic parameters, however, were differentially affected . While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently . The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY . Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride . MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not . Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites. J Biol Chem, 1996 Sep 27, 271(39), 24096 - 104 Functional dissection and site-directed mutagenesis of the structural gene for NAD(P)H-nitrite reductase in Neurospora crassa; Colandene JD et al.; Neurospora crassa NAD(P)H-nitrite reductase, encoded by the nit-6 gene, is a soluble, alpha2-type homodimeric protein composed of 127-kDa polypeptide subunits . This multicenter oxidation-reduction enzyme utilizes either NADH or NADPH as electron donor and possesses as prosthetic groups two iron-sulfur (Fe4S4) clusters, two siroheme groups, and two FAD molecules . The native activity of the enzyme is the NAD(P)H-dependent reduction of nitrite to ammonia . In addition, N . crassa nitrite reductase displays several partial activities in vitro, including a siroheme-independent NAD(P)H-cytochrome c reductase activity and an FAD-independent dithionite-nitrite reductase activity . These partial activities are presumed to be manifestations of discrete functional domains within the protein . A full-length nit-6 cDNA was constructed and used in developing an expression system within E . coli capable of yielding high levels of NADPH-nitrite reductase activity . Maximal expression was obtained in nirB- E . coli cells grown anaerobically at 22 +/- 1 degrees C, in conjunction with co-expression of a plasmid-borne cysG gene (encoding the rate-limiting enzyme in siroheme synthesis) and co-transformation with plasmid pGroESL (encoding bacterial chaperonins GroES and GroEL) . Dissection of gene segments encoding putative functional domains within the nit-6 gene was performed . Expression of a partial cDNA construct encoding the FAD-/NAD-binding domain yielded extracts with NADPH-cytochrome c reductase activity but no NADPH-nitrite reductase activity or dithionite-nitrite reductase activity . Expression of a cDNA construct encoding the (Fe4S4)-siroheme-binding domain resulted in extracts possessing dithionite-nitrite reductase activity but no NADPH-nitrite reductase or NADPH-cytochrome c reductase activity . Analysis of site-directed mutations altering amino acid residues Cys-331 within the FAD-/NAD-binding domain and Ser-755 within the (Fe4S4)-siroheme-binding domain of the nitrite reductase demonstrated that these residues were not essential for native or partial enzyme activity . Cys-757 within the (Fe4S4)-siroheme-binding domain was essential for native enzyme activity. J Biol Chem, 1996 Sep 27, 271(39), 24010 - 6 Kinetic characterization of dUTPase from Escherichia coli; Larsson G et al.; The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA . The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits . In the present work, the catalytic properties of the E . coli dUTPase were investigated in the pH range 5-11 . The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed) . The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10(5) times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP . With dUTP.Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s-1 . Km passed through a broad minimum in the neutral pH range with values approaching 10(-7) M . It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3 . When excess dUTPase was reacted with dUTP middle dotMg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step . The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10 . Only one enantiomer of the substrate analog 2'-deoxyuridine-5'-(alpha-thio)-triphosphate was hydrolyzed by the enzyme . These results are interpreted to favor a catalytic mechanism involving magnesium binding to the alpha-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products . The results are discussed with reference to recent data on the structure of the E . coli dUTPase.UDP complex. J Biol Chem, 1996 Sep 27, 271(39), 23954 - 9 Mechanism-based inactivation of dopa decarboxylase by serotonin; Bertoldi M et al.; Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit . In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia . These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis . The Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively . The NaBH4-reduced enzyme does not catalyze this reaction . Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min-1, respectively . Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine . Inactivation with {2-14C}5-HT results in the incorporation of 1 mol of label/enzyme subunit . Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21 . The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized . A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented. J Biol Chem, 1996 Sep 27, 271(39), 23884 - 94 Transcriptional pausing of RNA polymerase in the presence of guanosine tetraphosphate depends on the promoter and gene sequence; Krohn M et al.; We have studied the response of the effector molecule guanosine 3',5'-bisdiphosphate (ppGpp) on RNA polymerase pausing during in vitro transcription elongation . Pausing was followed during single round extension of stalled ternary complexes excluding possible ppGpp effects on initiation . The ppGpp dependences of early pausing sites within different transcription systems controlled by promoters with known response to enhanced ppGpp levels in vivo were quantitatively characterized . Transcription of stable RNAs and mRNA genes were analyzed . In addition, the in vitro pausing behavior of two promoter variants directing the same sequence but differing in their in vivo ppGpp sensitivity were compared . In the presence of ppGpp we noted a slight general enhancement of specific pauses in all transcription systems . However, genes known to be under stringent or growth rate control in vivo revealed a notably stronger pausing enhancement . The sites of pausing are not changed by the presence of ppGpp but appear to be sequence-specific . The effect of ppGpp on the extent of pausing depends on the particular promoter and closely adjacent sequences that the RNA polymerase has passed during initiation . Pausing enhancement requires the presence of ppGpp during elongation but not during initiation . The results underline the importance of pausing for transcription regulation and offer a plausible explanation for inhibition of stable RNA expression under conditions of elevated concentrations of ppGpp. J Biol Chem, 1996 Sep 27, 271(39), 23874 - 83 Interaction of Escherichia coli RecA protein with LexA repressor . II . Inhibition of DNA strand exchange by the uncleavable LexA S119A repressor argues that recombination and SOS induction are competitive processes; Harmon FG et al.; The Escherichia coli RecA protein is involved in SOS induction, DNA repair, and homologous recombination . In vitro, RecA protein serves as a co-protease to cleave LexA repressor, the repressor of the SOS regulon; in addition, RecA protein promotes homologous pairing and DNA strand exchange, steps important to homologous recombination and DNA repair . To determine if these two functions of RecA protein are competing or parallel, the effect of uncleavable LexA S119A repressor on RecA protein-dependent activities was examined . LexA S119A repressor inhibits both the single-stranded DNA (ssDNA)-dependent ATP hydrolysis and DNA strand exchange activities of RecA protein . As for wild-type LexA repressor (Rehrauer, W . M., Lavery, P . E., Palmer, E . L., Singh, R . N., and Kowalczykowski, S . C . (1996) J . Biol . Chem . 271, 23865-23873), inhibition of ATP hydrolysis is dependent upon the presence of E . coli single-stranded DNA binding (SSB) protein, arguing that LexA repressor affects the competition between RecA protein and SSB protein for ssDNA binding sites . In contrast, inhibition of DNA strand exchange activity is SSB protein-independent, suggesting that LexA S119A repressor blocks a site required for DNA strand exchange . These results imply that there is a common site on the RecA protein filament for secondary DNA and LexA repressor binding and raise the possibility that the recombination and co-protease activities of the RecA protein filament are competitive. J Biol Chem, 1996 Sep 27, 271(39), 23865 - 73 Interaction of Escherichia coli RecA protein with LexA repressor . I . LexA repressor cleavage is competitive with binding of a secondary DNA molecule; Rehrauer WM et al.; Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively . Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels . The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein . However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites . Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive . The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination. J Biol Chem, 1996 Sep 27, 271(39), 23615 - 8 High field EPR studies of mouse ribonucleotide reductase indicate hydrogen bonding of the tyrosyl radical; Schmidt PP et al.; Ribonucleotide reductase catalyzes by free radical chemistry the reduction of ribonucleotides to deoxyribonucleotides . The R2 protein of a class 1 ribonucleotide reductase contains a stable tyrosyl radical of neutral phenoxy character, which is necessary for normal enzymatic activity . Here we present the EPR spectra from the tyrosyl free radical in the R2 protein from mouse at 9.62, 115, and 245 GHz . We show that the g-value anisotropy of the mouse R2 radical, when precisely determined from high field EPR spectra, is similar to that of the hydrogen bonded dark stable YD middle dot tyrosyl radical of photosystem II and different from that of the Escherichia coli R2 radical . Because the g-value anisotropy is an important indicator of the hydrogen bonding status of the tyrosyl radical, this result suggests that the mouse R2 radical has its tyrosylate oxygen hydrogen bonded with a D2O exchangeable proton, whereas this hydrogen bond is absent in the E . coli enzyme . It is suggested that the observed proton may be derived from the tyrosine that will become a tyrosyl radical. Gene, 1996 Sep 26, 174(1), 51 - 8 Construction and expression of a synthetic wheat storage protein gene; Anderson OD et al.; A synthetic wheat high-molecular-weight (HMW) glutenin storage protein gene analog was constructed for expression in E . coli . This first synthetic HMW-glutenin gene and future modifications are intended to allow systematic dissection of the molecular basis of HMW-glutenin role in the visco-elastic properties critical for wheat product processing and utilization . The design of the gene included four features: different construction strategies for the separate assembly of major polypeptide domains, the inclusion of convenient restriction sites for modifications, use of a codon selection similar to E . coli highly expressed genes, and the ability to produce repetitive sequence domains of exact numbers of defined repeats . The complete synthetic HMW-glutenin construct was 1908 bp, and contained 32 identical copies of one of the HMW-glutenin repetitive domain motifs . The gene expressed the novel HMW-glutenin protein to relatively high levels in bacterial cultures and the protein exhibited the known anomalous behavior of HMW-glutenins in SDS-PAGE. Mol Gen Genet, 1996 Sep 25, 252(4), 398 - 403 Transcription-induced deletions in plasmid vectors: M13 DNA replication as a source of instability; Vilette D et al.; We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events . We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions . The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events . pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used . Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations . We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded . This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms . Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation . In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage. Biochem Biophys Res Commun, 1996 Sep 24, 226(3), 822 - 9 Mutations in a putative zinc-binding domain inactivate the mitochondrial intermediate peptidase; Chew A et al.; The mitochondrial intermediate peptidase (MIP) cleaves characteristic octapeptides, (F/L/I)XX(T/S/ G)XXXX(decreases), from the N-terminus of many imported mitochondrial proteins . This leader peptidase is activated by divalent cations and inactivated by thiol-blocking agents, properties which are typical of metallo- and cysteine-proteases, respectively . To elucidate the mechanism of action of MIP, we analyzed by site-directed mutagenesis the functional role of a putative zinc-binding domain (F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E) and two cysteine residues (C131 and C581), which are highly conserved in evolutionarily distant MIP sequences . We show that two histidines and a glutamic acid in the H-E-X-G-H motif and a glutamic acid 25 residues from the second histidine are essential for MIP function in vivo . In contrast, C131 and C581 are important for protein stability but are not required for activity in vivo or in vitro . These findings are consistent with MIP being a metallopeptidase. Biochemistry, 1996 Sep 24, 35(38), 12623 - 8 Seeding of A beta fibril formation is inhibited by all three isotypes of apolipoprotein E; Wood SJ et al.; Apolipoprotein E is immunochemically localized to amyloid plaque in Alzheimer's brains, and the allelic distribution of ApoE in individuals is associated with a disposition toward Alzheimer's disease . We show here that all three ApoE isotypes exhibit a strong and specific ability to inhibit both nucleation and seeding of fibril formation by the A beta peptide in vitro . A beta (1-40) depleted of aggregates requires long incubation times before the onset of fibril formation, but addition of very low levels of A beta fibrils to such reactions is sufficient to reduce or eliminate this lag time . ApoE added to such seeded reactions extends the lag time in a dose-dependent manner, so that higher levels of seeding require higher levels of ApoE to achieve a given delay time to reaction onset . This effect is observed with all three isotypes produced in Escherichia coli, as well as with plasma-derived ApoE and the N-terminal domain of ApoE3 produced in E . coli . In contrast, bovine serum albumin and the four-helix bundle protein interleukin-4 are poor inhibitors of seeding . ApoE3 can also inhibit fibril formation by A beta (1-42) . The three full-length isotypes of ApoE produced in E . coli are equipotent at inhibition . It is therefore possible that the genetics of ApoE and AD may fundamentally depend on the ability of ApoE to inhibit seeding but that the trends in the genetics must be related to something other than the specific activities of the native ApoE isoforms used in these studies . The data show ApoE to be the first member of a new class of fibril formation inhibitor that acts by blocking the seeding of fibril growth. Biochemistry, 1996 Sep 24, 35(38), 12526 - 31 Truncations of the C-terminus have different effects on the conformation and activity of phosphatidylinositol transfer protein; Voziyan PA et al.; Contributions of the C-terminus toward the conformation and activity of phosphatidylinositol transfer protein (PITP) were studied by comparing properties of the 271 amino acid, full-length protein, PITP(1-271), and two truncated species, PITP(1-259) and PITP(1-253) . Using recombinant proteins and an in vitro phospholipid transfer assay with phosphatidylcholine vesicles, the activities of PITP(1-271) and PITP(1-259) were identical, while the activity of PITP(1-253) was almost totally abolished . By most physical and chemical criteria, however, PITP(1-259) and PITP(1-253) were virtually indistinguishable and differed significantly from the full-length protein . Results of second derivative analysis of absorbance spectra were consistent with an additional two Tyr residues being exposed to the solvent in PITP(1-259) and PITP(1-253) in comparison to PITP(1-271) . Only one out of four Cys residues in PITP(1-271) reacted with dithiobisnitrobenzoic acid, while two Cys residues were accessible in both truncated species . Quenching of intrinsic Trp fluorescence by acrylamide demonstrated an increase in exposure of Trp residues in both PITP(1-259) and PITP(1-253); binding of the fluorescence probe 1,8-ANS to these proteins was also significantly higher compared to PITP(1-271) . These results describe a more relaxed overall tertiary structure brought about by the C-terminal truncations . This altered structure did not affect the stability of the truncated proteins, as indicated by equilibrium unfolding in guanidinium chloride . Refolding of the denatured PITP(1-259), however, was considerably slower than that of full-length PITP . Our study suggests a critical role of the C-terminal residues 254-259 in transfer activity of PITP . Residues 260-271, on the other hand, appear to be more important for the rapid folding and maintenance of a compact native conformation of the protein. Biochemistry, 1996 Sep 24, 35(38), 12503 - 10 Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini; Liu J et al.; The solution structure and internal dynamics of the reactive-site (Lys44-Asp45 peptide bond) hydrolyzed form of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V*) were characterized by the application of two-dimensional 1H-15N NMR methods to the uniformly 15N-labeled protein . The 1H-15N chemical shift correlation spectra of rCMTI-V* were assigned, and the chemical shift data were compared with those available for rCMTI-V {Liu, J., Prakash, O., Cai, M., Gong, Y., Huang, Y., Wen, L., Wen, J . J., Huang, J.-K., & Krishnamoorthi, R . (1996) Biochemistry 35, 1516-1524} and CMTI-V* {Cai, M., Gong, Y., Prakash, O., & Krishnamoorthi, R . (1995) Biochemistry 34, 12087-12094} for which three-dimensional solution structures have been determined . It was deduced that the solution structure of rCMTI-V* was almost the same as that of CMTI-V* . 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and inverted question mark1H inverted question mark-15N steady-state heteronuclear Overhauser effects were measured for the peptide NH units and arginine and tryptophan N epsilon H groups in rCMTI-V*, and the model-free parameters {Lipari, G., & Szabo, A . (1982) J . Am . Chem . Soc . 104, 4546-4559, 4559-4570} were computed . Most of the backbone of rCMTI-V* is found to be highly constrained (S2 = 0.85), including the N-terminal residues 3-6 (S2 = 0.77) . Residues 39-44, forming the C-terminal fragment of the binding loop, exhibit increased mobility (S2 = 0.51); however, the N-terminal segment (residues 46-48) retains rigidity as in the intact form (S2 = 0.83) . The S2 values, 0.78 and 0.59, respectively, of Arg50 and Arg52 side chain NHs provide evidence not only for the conservation of the Arg hydrogen-bonds with the binding loop segments but also for the difference in strength between them . This is consistent with the earlier observation made from a study of rCMTI-V at two different pHs and its R50 and R52 mutants {Cai, M., Huang, Y., Prakash, O., Wen, L., Dunkelbarger, S . P., Huang, J.-K., Liu, J., & Krishnamoorthi, R . (1996) Biochemistry 35, 4784-4794} . The dynamical results suggest the mainchain oxygen atom of Asp45 as the hydrogen bond acceptor of Arg50 . Residues Trp9 and Trp54, which interact with many others in the protein scaffold and the binding loop region, respectively, remain rigid in the cleaved inhibitor with the S2 values of 0.84 and 0.71 determined for their respective N epsilon Hs . The internal dynamics of rCMTI-V* was compared with that of the noncovalent complex formed between the two fragments of reactive-site-hydrolyzed chymotrypsin inhibitor-2 from barley seeds {CI-2; Shaw, G . L., Davis, B., Keeler, J., & Fersht, A . R . (1995) Biochemistry 34, 2225-2233}, another potato I family inhibitor that lacks the Cys3-Cys48 disulfide present in rCMTI-V*. Biochemistry, 1996 Sep 24, 35(38), 12487 - 94 Deacylation and reacylation for a series of acyl cysteine proteases, including acyl groups derived from novel chromophoric substrates; Doran JD et al.; In order to investigate structure-reactivity relationships within a series of acyl cysteine proteases {Doran, J . D., & Carey, P . R . (1996) Biochemistry 35, 12495-12502}, deacylation kinetics have been measured for a number of acyl intermediates involving members of the papain superfamily . Derivatives of the "simple" chromophoric ligand (5-methylthienyl)acrylate (5MTA) and those based on two chromophorically labeled derivatives of peptidyl substrates, viz., 2-{(N-acetyl-L-phenylalanyl)amino}-3-(5-methylthienyl)acrylate (Phe5MTA) and 2-{(N-acetyl-L-alanyl)amino}-3-(5-methylthienyl)acrylate (Ala5MTA), were used to create acyl enzyme adducts with papain, cathepsin B, and cathepsin L . The chromophoric specific substrates were designed to utilize hydrogen-bonding and hydrophobic interactions which are known to be important in promoting catalysis by papain . For cathepsins B and L, removing one of the hydrogen-bonding donors making up the putative oxyanion hole retards deacylation by 3-25-fold, demonstrating that the oxyanion hole has a modest effect on catalysis for these substrates . With the above substrates and the wild-type and oxyanion hole mutants, the values of the deacylation rate constants stretch over a 214-fold range, from 0.07 to 15 x 10(-3) s-1 . The pKa for deacylation of {(5-methylthienyl)-acryloyl}papain is 4.9, close to that reported for similar papain intermediates, while that for Ala5MTA-papain is at 3.5, which in the latter likely represents the effect of the P1-S1 and P2-S2 interactions on the environment of histidine-159 . For the Phe5MTA-papain the extent of deacylation was found to depend on the pH and the starting acyl enzyme concentration . A simple model has been derived which accounts quantitatively for this behavior, using the assumptions that the protonated form of the acyl product reacylates the enzyme and that in the pH range 5.0-7.5 the ionization of active-site groups has no effect on reacylation . The validity of the first assumption was demonstrated by following the deacylation of Phe5MTA-papain in the presence of the potent inhibitor E-64 {1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane}, whereupon complete deacylation occurred at all pHs with a pKa identical to that for Ala5MTA-papain, viz., 3.5. Biochemistry, 1996 Sep 24, 35(38), 12479 - 86 Factors influencing redox thermodynamics and electron self-exchange for the {Fe4S4} cluster in Chromatium vinosum high potential iron protein: the role of core aromatic residues in defining cluster redox chemistry; Soriano A et al.; The roles of aromatic core residues in regulating the reduction potential, the enthalpy and entropy of reduction, and the self-exchange rate constants for electron-transfer reactions for the prosthetic {Fe4S4}3+/2+ cluster of Chromatium vinosum high potential iron protein (HiPIP) have been addressed by a combination of site-directed mutagenesis, high field NMR (EXSY) experiments, and variable temperature spectrochemical redox titration measurements . Minimal changes are observed following nonconservative mutation of residues Tyr19, Phe48, and Phe66 . Apparently these hydrophobic residues play only a minor role in defining the electronic properties of the cluster . These data support a model, first defined from results obtained on Tyr19 mutant HiPIP's {Agarwal, A., Li, D., & Cowan, J.A . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 9440-9444}, in which the aromatic core restricts solvent accessibility and thereby stabilizes the oxidized {Fe4S4}3+ cluster. Biochemistry, 1996 Sep 24, 35(38), 12455 - 63 Electrochemical measurement of second-order electron transfer rate constants for the reaction between cytochrome b5 and cytochrome c; Seetharaman R et al.; The second-order electron transfer reaction between cytochrome b5 and cytochrome c has been studied by cyclic voltammetry utilizing a gold electrode modified with beta-mercaptopropionate . When cyclic voltammetry is performed on a solution containing a mixture of cytochrome b5, cytochrome c and polylysine, cytochrome b5 undergoes reversible electrochemistry at the electrode surface while cytochrome c discriminates against the electrode surface . The selectivity of the modified electrode for negatively charged proteins makes it possible to selectively reduce a protein possessing a net negative charge and a relatively low reduction potential (outer mitochondrial membrane cytochrome b5, Eo = -102 mV; microsomal cytochrome b5, Eo = 3 mV) in the presence of another protein possessing a net positive charge and a relatively high reduction potential (cytochrome c, Eo = 265 mV) . The electrochemical reduction of ferricytochrome b5 at the electrode surface is followed by a second-order electron transfer reaction between ferrocytochrome b5 and ferricytochrome c that yields ferricytochrome b5 and ferrocytochrome c . This fast homogeneous electron transfer reaction which is preceded by a heterogeneous electron transfer reaction results in a characteristic cyclic voltammogram containing a pre-peak to the reduction current . The second-order rate constant for the homogeneous reaction was obtained by invoking the above reaction scheme for digital simulation of a cyclic voltammogram which was subsequently fitted to the experimental data . Second-order rate constants obtained with this method are 2.9 x 10(8) and 8.9 x 10(8) for the homogeneous electron transfer reactions between rat liver outer mitochondrial membrane (OM) ferrocytochrome b5 and beef liver microsomal ferrocytochrome b5, with horse heart ferricytochrome c, respectively . These values are in good agreement with second-order rate constants obtained for the same protein systems by flash photolysis . {Meyer, T . E., Rivera, M., Walker, F . A., Mauk, M . R., Mauk, A . G., Cusanovich, M . A., & Tollin, G . (1993) Biochemistry 32, 622-627}. Biochemistry, 1996 Sep 24, 35(38), 12402 - 11 Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase; Nandy A et al.; The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH) . In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G . These glutamates thus act on substrate approaching from two opposite regions at the active center . We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu . The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively . MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA . This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA) . The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376 . The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower . The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases . The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper {Lee et al . (1996) Biochemistry 35, 12412-12420}. Biochemistry, 1996 Sep 24, 35(38), 12387 - 401 Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z) . 4 . Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases; Richard JP et al.; Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined . The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1 . By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate . These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion . We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461 . The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme . However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose . These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates . The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water . This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents. Biochemistry, 1996 Sep 24, 35(38), 12377 - 86 Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z) . 3 . Evidence that Glu-461 participates in Brønsted acid-base catalysis of beta-D-galactopyranosyl group transfer; Richard JP et al.; Experiments are reported to determine the role of Glu-461 in the beta-D-galactopyranosyl group transfer reaction catalyzed by beta-galactosidase . E461G beta-galactosidase catalyzes the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside through a galactosyl-enzyme intermediate that shows a high reactivity toward the anionic nucleophile azide ion, but no detectable reactivity toward the neutral nucleophile trifluoroethanol . By contrast, the galactosylated wild type enzyme is reactive toward trifluoroethanol but not anions . The change in specificity observed for the E461G mutant can be rationalized by a mechanism in which Glu-461 participates in general acid-base catalysis at the leaving group/nucleophile . The observed low activity of E461G beta-galactosidase for hydrolysis of 2,2,2-trifluoroethyl beta-D-galactopyranoside is due entirely to a wild type enzyme contaminant in our preparation of the mutant enzyme, and the mutant enzyme itself has essentially no catalytic activity for cleavage of this substrate . The substitution of glutamate at position 461 by glycine leads to a more than 500 000-fold reduction in the rate constant for enzymatic cleavage of the glycosidic bond to the strongly basic trifluoroethoxide leaving group (pKa = 12.4), but to a smaller 1300-fold reduction in the rate constant for cleavage of the bond to the more weakly basic 4-nitrophenoxide leaving group (pKa = 7.1) . This corresponds to a more than 3.5 kcal/mol greater stabilization by Glu-461 of the transition state for the reaction of the substrate with the more basic trifluoroethoxide leaving group . These data are consistent with the conclusion that Glu-461 provides general acid catalysis of leaving group departure, which is most effective for cleavage of the relatively strong bonds to basic alkoxide leaving groups. Biochemistry, 1996 Sep 24, 35(38), 12369 - 76 Trichodiene synthase . Probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis; Cane DE et al.; Trichodiene synthase catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon trichodiene . The enzyme normally requires a divalent cation, Mg2+, which can be substituted by Mn2+ . Trichodiene synthase from Fusarium sporotrichioides has a highly conserved aspartate rich region, aa 100-104 (DDSKD) . Three mutants were constructed by site-directed mutagenesis in which each aspartate residue was individually replaced by glutamate . The mutants were each overexpressed and purified to homogeneity . The importance of Asp100 and Asp101 for catalysis was established by the observation of an increase in Km as well as a reduction in kcat in the corresponding Glu mutants . Replacement of the Asp104 residue with Glu had little effect on either Km or kcat . All three mutants produced anomalous sesquiterpene products in addition to trichodiene when incubated with farnesyl diphosphate . Interestingly, when Mg2+ was replaced by Mn2+ in the incubation buffer, the kcat/Km of both wild type trichodiene synthase and the D104E dropped significantly, while those of the other two mutants were not much affected . The proportion of anomalous products increased significantly when the D100E and D101E mutants were incubated in the presence of Mn2+ . These observations all lend weight to the proposal that the aspartate residues mediate substrate binding by chelation of the divalent metal ion . Asp100 and Asp101 appear to play a relatively more important role than Asp104. Biochemistry, 1996 Sep 24, 35(38), 12354 - 62 Cleavage of DNA by the insulin-mimetic compound, NH4{VO(O2)2(phen)}; Hiort C et al.; The kinetics and mechanism of cleavage of DNA by the insulin-mimetic peroxo-vanadate NH4{VO(O2)2(phen)}, pV, are described . In the presence of low energy UV radiation or biologically common reducing agents, pV decomposes into the monomer, dimer, and tetramer of vanadate and an uncharacterized compound of V4+ as shown by 51V NMR, ESR, and absorption spectra . The rate of photodecomposition of pV is reduced in the presence of calf thymus DNA, indicating that a decomposition product of the peroxo-vanadate, that is important in the destruction pathway of the complex, is interacting with DNA . This species, probably a short-lived complex of V4+, may also be responsible for the observed catalytic decomposition of pV in the absence of DNA by ascorbate . If closed circular pBR322 DNA is present when the peroxo-vanadate is destroyed by either UV radiation or reducing agents, the polymer may have its sugar-phosphate backbone broken . Closed circular DNA (form I) is converted into nicked circular DNA (form II) and linear DNA (form III) . The amounts of the various forms produced as a function of irradiation time and peroxo-vanadate concentration were fit to a kinetic model to derive rate constants for the conversions . The kinetic analysis shows that pV is a single-strand nicking agent which exhibits some base and/or sequence preference . Furthermore, the pH dependences of the rates for conversion of form I to form II and for conversion of form II to form III are different, indicating that the nature of the chemistry at the site of cleavage on DNA influences further cutting by activated pV . Reduced amounts of DNA breakage in the presence of various salts and metal binding ligands indicate that a short-lived reactive complex of V4+, not the V4+ species detected by ESR at long irradiation times, is important in the cleavage process . The susceptibility of pV to decomposition by biologically common reducing agents suggests that metabolites of the agent, and not the compound itself, are responsible for its insulin-mimetic effects. Biochemistry, 1996 Sep 24, 35(38), 12228 - 34 Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme; Gonzalez JC et al.; Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine . Previous work had shown the existence of a reactive thiol group, cysteine 726, whose alkylation led to loss of all detectable enzymatic activity {Gonzalez, J.C., et al . (1992) Biochemistry 31, 6045-6056} . A site-directed mutation of MetE, Cys726Ser, was constructed to investigate the possible role of this cysteine . The Cys726Ser protein was purified to homogeneity, affording a protein with no detectable activity . To assess the possibility that cysteine726 functions as a metal ligand, inductively coupled plasma-atomic emission spectrometry was performed . The wild-type enzyme contains 1.02 equiv of zinc per subunit; the Cys726Ser mutant does not contain zinc, supporting the view that cysteine726 is required for metal binding . A loss of enzymatic activity is observed upon removal of zinc from the wild-type MetE by incubation in urea and EDTA; activity can subsequently be restored by zinc reconstitution, suggesting that zinc is required for catalysis . Circular dichroism measurements further suggest that there are no major differences in the secondary structures of the wild-type and the Cys726Ser mutant enzymes . Extended X-ray absorption fine structure analysis has established that the average zinc environment is different in the presence of homocysteine than in its absence and is consistent with the changes expected for displacement of an oxygen or nitrogen ligand by the sulfur of homocysteine . A possible model for zinc-dependent activation of homocysteine by MetE is presented. FEBS Lett, 1996 Sep 23, 394(1), 71 - 5 5S rRNA sugar-phosphate backbone protection in complexes with specific ribosomal proteins; Shpanchenko OV et al.; 5S ribosomal RNA forms stable specific complexes with ribosomal proteins L18, L25 and L5 . In this work, interaction of phosphate residues of E . coli 5S rRNA within 5S rRNA-protein complexes has been studied . For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied . In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA . This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized . In the 5S rRNA-L18 complex only two phosphate groups, G7 and A34, were protected . L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups concentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA . The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure. FEBS Lett, 1996 Sep 23, 394(1), 66 - 70 Unusual enzyme characteristics of aspartyl-tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp . KOD1; Fujiwara S et al.; The aspA gene, encoding the aspartyl-tRNA synthetase (AspRS) from the hyperthermophilic archaeon Pyrococcus sp . KOD1, was expressed in Escherichia coli . The KOD1 AspRS, which was purified to homogeneity and was shown to be functional in dimeric form, aminoacylated tRNA from KOD1 . The optimum temperature for this activity was 65 degrees C, which was lower than that for the cell growth of KOD1 (85 degrees C) . However, it increased to 75 degrees C by the addition of polyamine molecules, such as putrescine, spermine, and spermidine . Analysis of the thermal denaturations of the enzyme and of KOD1-tRNA indicated that neither of them was denatured at temperatures below 70 degrees C . These results suggest polyamine is one of the factors which are required to stabilize the AspRS-tRNA complex in vivo . In order to determine whether the nucleotide triphosphate (NTP) is required for Asp-tRNA synthesis, the aminoacylation was examined in the presence of each of the four NTPs . AspRS most effectively aminoacylated tRNA in the presence of ATP . However, we also found that the enzyme aminoacylated it even in the presence of GTP and UTP as well . Archaeon synthetase may have an interesting system to utilize other NTPs than ATP . The extreme conditions of early life may have given rise to these unique characteristics which then disappeared from developed organisms through evolution. Carbohydr Res, 1996 Sep 23, 291, 127 - 39 Structural studies of the enteroinvasive Escherichia coli (EIEC) O28 O-antigenic polysaccharide; Rundlof T et al.; The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O28 has been investigated . NMR spectroscopy has been the main method used, complemented with sugar and methylation analyses . The polysaccharide contains one equivalent of O-acetyl groups per repeating unit . Selective cleavage of the O-deacetylated polymer was performed by treatment with aqueous hydrofluoric acid, and resulted in a trisaccharide-glycerol . The polysaccharide thus is of the teichoic acid type and composed of repeating units in which the trisaccharide-glycerol residues are joined by phosphodiester linkages . The O-antigen polysaccharide has the following structure . {sequence: see text} The absolute configuration of the glycerol moiety as R, )i.e., D-glycerol 1-phosphate) was determined by a new method based on TEMPO oxidation of the polysaccharide, followed by GLC analysis of the (+)-2-butyl ester of the resulting glyceric acid. Carbohydr Res, 1996 Sep 23, 291, 43 - 52 Evaluation of C-(beta-D-galactosyl) and C-(2-deoxy-D-lyxo-hex-1-enopyranosyl) (D-galactal type) derivatives as inhibitors of beta-D-galactosidase from Escherichia coli; Kiss L et al.; C-(2-Deoxy-D-lyxo-hex-1-enopyranosyl)formamide was prepared from acetylated C-(beta-D-galactopyranosyl)formamide by a radical-mediated bromination-zinc/N-methylimidazole-induced reductive elimination-Zemplen deacetylation reaction sequence . The preparation of acetylated 5-(2-deoxy-D-lyxo-hex-1-enopyranosyl)tetrazole was improved . Methyl C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formimidate was transformed by benzylamine into N-benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamidine and, after hydrolysis to methyl C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formate, into N-benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamide . A series of C-(beta-D-galactopyranosyl) and C-(2-deoxy-D-lyxo-hex-1-enopyranosyl) derivatives was comparatively investigated for E . coli beta-D-galactosidase inhibitory activity . N-Benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamidine was the best inhibitor and had K(i) = 6 microM (on the basis of its free base concentration, K(i) = 8.3 nM was obtained) . Basicity and hydrophobicity of the aglycon proved to be more important factors for the inhibition than the conformation of the sugar ring. Mutat Res, 1996 Sep 23, 356(2), 237 - 45 Nitro musks are cogenotoxicants by inducing toxifying enzymes in the rat; Mersch-Sundermann V et al.; In the present study, musk xylene (MX) and musk ketone (MK) were examined for their potency to induce toxifying enzymes in the liver of Sprague-Dawley rats, using an in vivo/in vitro model . After i.p . application of 10, 20 and 40 mg/day MX and MK over a period of 5 days, 9000 x g liver fractions (S9M) were used to study the toxification of a number of well-known pregenotoxicants in the SOS chromotest, i.e., benzo{a}pyrene (B{a}P), 2-aminoanthracene (2-AA), and aflatoxin B1 (AFB1) . The genotoxic potencies of B{a}P, 2-AA and AFB1 in the presence of S9M were compared to those obtained in the presence of S9 fractions of untreated animals (S9O, negative control) . S9M fractions derived from MK-treated rats showed an increased potency to toxify B{a}P, 2-AA and AFB1 in comparison to S9O fractions (for instance: TIP{toxifying induction potency} = 70 per nmol AFB1 using 10 mg MK treatment) . In comparison, S9M fractions from MX-pretreated rats exhibited an increased genotoxicity only when using 2-AA (TIP = 0.04) and AFB1 (TIP = 61) as pregenotoxicants, but not when using B{a}P . To summarize the results, both MX and MK were strong inducers of toxifying liver enzymes . Therefore, these compounds seem to be cogenotoxicants for a number of well-known pregenotoxicants . Synergistic effects were found when using inducers of toxifying enzymes and pregenotoxicants in the in vivo/in vitro induction model. Mutat Res, 1996 Sep 23, 356(2), 229 - 35 Induction of SOS-independent mutations by benzo{a}pyrene treatment in Escherichia coli cells deficient in MutY or MutM DNA glycosylases: possible role of oxidative lesions; Urios A et al.; The induction of SOS-independent mutations by exposure to benzo{a}pyrene (BaP) was screened in Escherichia coli strains lacking SOS mutagenesis proteins and deficient in MutY or MutM glycosylases, which prevent mutations by 8-hydroxyguanine (GO lesion) . Mutagenicity assays, performed in the presence of S9 mix, indicated a great increase in the reversion of the trpE65 ochre mutation in both mutY and mutY mutM strains, whereas a lower increase was observed in a mutM strain . This mutability by BaP was observed in either uvr- or uvr+ strains . Moreover, it was increased when strains carried a deletion of the oxyR gene that abolished the OxyR response to oxidative stress, and reduced in the presence of the oxyR2 allele that rendered constitutive such response . It is suggested that SOS-independent mutations in cells treated with BaP arise from adenine/GO mispairs . The interaction of radical scavengers with BaP ultimate metabolites could cause oxidative stress capable of producing GO lesions . Strains lacking mutagenesis proteins and deficient in base excision repair systems, such as those dependent on MutY and MutM glycosylases, could be useful for screening the induction of SOS-independent mutations. J Chromatogr B Biomed Appl, 1996 Sep 20, 684(1-2), 147 - 61 Purification methods of mammalian catechol-O-methyltransferases; Tilgmann C et al.; The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COMT) polypeptides are reviewed . Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculevirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail . The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail . The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors. J Mol Biol, 1996 Sep 20, 262(2), 225 - 42 The crystal structure of glutamine-binding protein from Escherichia coli; Hsiao CD et al.; The crystal structure of the glutamine-binding protein (GlnBP) from Escherichia coli in a ligand-free "open" conformational state has been determined by isomorphous replacement methods and refined to an R-value of 21.4% at 2.3 A resolution . There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry . The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules . The structure has root-mean-square deviations of 0.013 A from "ideal" bond lengths and 1.5 degrees from "ideal" bond angles . The GlnBP molecule has overall dimensions of approximately 60 A x 40 A x 35 A and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel beta-strands . The small domain contains three alpha-helices and four parallel and one antiparallel beta-strands . The large domain is similar to the small domain but contains two additional alpha-helices and three more short antiparallel beta-strands . A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed . A model of the "closed form" GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and "open form" GlnBP . This model has been successfully used as a search model in the crystal structure determination of the "closed form" GlnBP-Gln complex by molecular replacement methods . The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 A . Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein . The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described. J Mol Biol, 1996 Sep 20, 262(2), 202 - 24 Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A; Clausen T et al.; Cystathionine beta-lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C beta-S bonds of a broad variety of substrates . The crystal structure of CBL from E . coli has been solved using MIR phases in combination with density modification . The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data . The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry . A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry . Each monomer of CBL can be described in terms of three spatially and functionally different domains . The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one beta-strand . It contributes to tetramer formation and is part of the active site of the adjacent subunit . The second domain (residues 61 to 256) harbors PLP and has an alpha/beta-structure with a seven-stranded beta-sheet as the central part . The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded beta-sheet . The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases . Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway . Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes . The structure of the beta, beta, beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2% . It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma. J Mol Biol, 1996 Sep 20, 262(2), 151 - 67 Structural analysis of Sindbis virus capsid mutants involving assembly and catalysis; Choi HK et al.; Sindbis virus core protein (SCP) has been isolated from virus and crystallized . The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure . The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed in Escherichia coli . Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein . There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein . Hence, E . coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA . The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein . The monomer-monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions . Removal of the hydrogen bonds by single substitutions did not prevent dimer formation . However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation . The wild-type truncated SCP is active in E . coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264 . Unlike the virus-extracted capsid protein, the E . coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis . Nevertheless, the E . coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket . Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues . These may represent successive steps of binding substrate prior to catalytic cleavage . Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules . Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166 . It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins . The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine . These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry. J Mol Biol, 1996 Sep 20, 262(2), 140 - 50 Probing the structural role of an alpha beta loop of maltose-binding protein by mutagenesis: heat-shock induction by loop variants of the maltose-binding protein that form periplasmic inclusion bodies; Betton JM et al.; The maltose-binding protein (MBP) of Escherichia coli is the periplasmic receptor of the maltose transport system . Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding . To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized . The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the protein . By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies . Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions . We found that the extent of formation of inclusion bodies in the periplasm of E . coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24. J Mol Biol, 1996 Sep 20, 262(2), 87 - 104 A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core; Myers CA et al.; The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA . The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain) . To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain . Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7 . CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site . Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees . The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain . The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron . Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site. J Mol Biol, 1996 Sep 20, 262(2), 77 - 86 The "+70 pause": hypothesis of a translational control of membrane protein assembly; Kepes F; Sequences of 66 genes encoding bacterial or yeast membrane proteins have been examined for the respective positioning of putative transmembrane domains and translational pauses . The latter were operationally defined as clusters of at least 17 non-preferred codons along the mRNA . The putative transmembrane domains were defined as stretches of at least 17 hydrophobic amino acids in the encoded protein . For yeast non-mitochondrial membrane proteins, it was observed that clusters of non-preferred codons occur more frequently about 56 to 75 codons after a hydrophobic stretch in the encoded protein . About 40 amino acid residues are required to span the large ribosomal subunit . Such clusters were thus predicted to cause a severe slow-down in peptide elongation, just when the hydrophobic stretch fully protrudes from the ribosome . This transient slow-down of the ribosome pace has consequently been named the "+70 pause" . This pause was not observed for mitochondrial or bacterial membrane proteins, which are thought to insert post-translationally in their respective membranes . Because insertion of yeast proteins in the endoplasmic reticulum membrane is generally cotranslational instead, it is possible that the "+70 pause" reflects the coupling of translation, targeting, insertion and folding in this case . The pause may, for instance, give time for productive interaction of the newly synthesized hydrophobic domain with the proper targeting/insertion machineries . Thus, it would favor entrance of the stalled protein domain into the proper pathway. J Mol Biol, 1996 Sep 20, 262(2), 71 - 6 Molecular symmetry of the ClpP component of the ATP-dependent Clp protease, an Escherichia coli homolog of 20 S proteasome; Shin DH et al.; The ClpP component Clp protease from Escherichia coli has been crystallized and examined by X-ray crystallography and self-rotation function calculations . The crystal belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 196.9 A, b = 104.3 A, c = 162.4 A and beta = 98.3 degrees . The X-ray diffraction pattern extends at least to 2.5 A Bragg spacing when exposed to CuK alpha X-rays . Self-rotation function analyses indicate that the ClpP oligomer has 72-point group symmetry . This symmetry suggests that the ClpP oligomer is a tetradecamer, (ClpP)14, consisting of two heptamers, (ClpP)7 stacked on top of each other in a head-to-head fashion . The measurement of crystal density indicates that two independent copies of the ClpP oligomers are present in the asymmetric unit, giving a crystal volume per protein mass (VM) of 2.73 A3/Da and a solvent content of 54.9% (v/v) . Self-rotation function calculations are consistent with the presence of two ClpP tetradecamers in the asymmetric unit . The Patterson function suggests that a translation of x = 0.5 and y = 0.5 relates a pair of ClpP oligomers in one asymmetric unit to another pair in the other asymmetric unit . And the two independent tetradecamers in one asymmetric unit are related by a relative rotation of about 18 degrees around the 7-fold axis. Cell, 1996 Sep 20, 86(6), 877 - 86 Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication; Yuzhakov A et al.; The E . coli replicase, DNA polymerase III holoenzyme, contains two polymerases for replication of duplex DNA . The DNA strands are antiparallel requiring different modes of replicating the two strands: one is continuous (leading) while the other is discontinuous (lagging) . The two polymerases within holoenzyme are generally thought to have asymmetric functions for replication of these two strands . This report finds that the two polymerases have equal properties, both are capable of replicating the more difficult lagging strand . Asymmetric action is, however, imposed by the helicase that encircles the lagging strand . The helicase contact defines the leading polymerase constraining it to a subset of actions, while leaving the other to cycle on the lagging strand . The symmetric actions of the two polymerases free holoenzyme to assemble into the replisome in either orientation without concern for a correct match to one or the other strand. J Biol Chem, 1996 Sep 20, 271(38), 23235 - 8 The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase . Site-directed mutagenesis of effector binding site residues; Al-Rabiee R et al.; D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli catalyzes the first committed step in serine biosynthesis and is allosterically regulated by L-serine, the end product of the pathway . Each subunit of the homotetramer is made up of three distinct domains with one of the intersubunit contacts being between adjacent regulatory domains . Each regulatory domain interface contains two symmetrical serine binding sites such that serine forms hydrogen bonds to both domains across the interface . Previous work (Al-Rabiee, R., Lee, E . J., and Grant, G . A . (1996) J . Biol . Chem . 271, 13013-13017) demonstrated that when adjacent regulatory domains are covalently linked to one another by engineered disulfide bonds, the enzyme was inactivated . Breaking the disulfide bonds by reduction restored enzymatic activity . This study demonstrates that the complementary situation is also true . Site-directed mutagenesis of three residues at the effector binding site, His344, Asn346, and Asn364', render the enzyme increasingly less susceptible to inhibition by the effector . When mutations result in a situation where it is no longer possible to establish a stable hydrogen bonding network across the regulatory domain interface, the inhibitory capacity of the effector is lost . Furthermore, mutations that produce as much as 5 orders of magnitude decrease in the ability of L-serine to inhibit the enzyme have no appreciable effect on the Km or kcat of the enzyme . These observations support the model that predicts that catalytic activity in D-3-phosphoglycerate dehydrogenase is regulated by the movement of adjacent regulatory domains about a flexible hinge and that effector binding tethers the regulatory domains together producing a state that results in a stable, open active site cleft that is no longer able to promote catalysis. Nature, 1996 Sep 19, 383(6597), 279 - 82 Three-dimensional structure of human cytomegalovirus protease; Shieh HS et al.; Herpesviruses encode a serine protease that specifically cleaves assembly protein . This protease is critical for replication, and represents a new target for antiviral drug design . Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution . The structure reveals a unique fold and new catalytic strategy for cleavage . The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin . The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157 . Dimerization, which seems to be necessary for activity, is observed in the crystals . Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding. Nature, 1996 Sep 19, 383(6597), 272 - 5 A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease; Tong L et al.; Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns . hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor . hCMV protease is a serine protease, although it has little homology to other clans of serine proteases . Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold . Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies . The structure suggests that the third member of the triad is probably His 157 . A dimer of the protease with an extensive interface is found in the crystal structure . This structure information will help in the design and optimization of inhibitors against herpesvirus proteases. Nature, 1996 Sep 19, 383(6597), 266 - 9 GTPase activity of Rab5 acts as a timer for endocytic membrane fusion; Rybin V et al.; The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport . Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein . To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of {alpha(32)P}-XTP hydrolysis in situ during endosome fusion in vitro . Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis . Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5 . Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10291 - 6 Regulation of SOS mutagenesis by proteolysis; Frank EG et al.; DNA damage-inducible mutagenesis in Escherichia coli is largely dependent upon the activity of the UmuD (UmuD') and UmuC proteins . The intracellular level of these proteins is tightly regulated at both the transcriptional and the posttranslational levels . Such regulation presumably allows cells to deal with DNA damage via error-free repair pathways before being committed to error-prone pathways . We have recently discovered that as part of this elaborate regulation, both the UmuD and the UmuC proteins are rapidly degraded in vivo . We report here that the enzyme responsible for their degradation is the ATP-dependent serine protease, Lon . In contrast, UmuD' (the posttranslational product and mutagenically active form of UmuD) is degraded at a much reduced rate by Lon, but is instead rapidly degraded by another ATP-dependent protease, ClpXP . Interestingly, UmuD' is rapidly degraded by ClpXP only when it is in a heterodimeric complex with UmuD . Formation of UmuD/UmuD' heterodimers in preference to UmuD' homodimers therefore targets UmuD' protein for proteolysis . Such a mechanism allows cells to reduce the intracellular levels of the mutagenically active Umu proteins and thereby return to a resting state once error-prone DNA repair has occurred . The apparent half-life of the heterodimeric UmuD/D' complex is greatly increased in the clpX::Kan and clpP::Kan strains and these strains are correspondingly rendered virtually UV non-mutable . We believe that these phenotypes are consistent with the suggestion that while the UmuD/D' heterodimer is mutagenically inactive, it still retains the ability to interact with UmuC, and thereby precludes the formation of the mutagenically active UmuD'2C complex. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10228 - 33 Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells; Eldar-Finkelman H et al.; In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity . The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA . Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT . Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants . Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells) . Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue . The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity . A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant . Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively) . These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10134 - 8 Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro; Yao R et al.; A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence . The cDNA encodes a 318-amino acid protein of Mr 35,960 . The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase . In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence . There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme . Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates . With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product . The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate . Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity . Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate . These properties are consistent with the enzymes derived from human and rat sources. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10128 - 33 Interaction between human tRNA synthetases involves repeated sequence elements; Rho SB et al.; Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex . Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system . Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix . This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library . The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex . Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively . Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs . Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex . The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10123 - 7 Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli; Wu J et al.; Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII . To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label . Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII . Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228 . Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs . The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A . Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A . long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226 . The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10094 - 8 SoxR, a {2Fe-2S} transcription factor, is active only in its oxidized form; Gaudu P et al.; SoxR protein is known to function both as a sensor and as a transcriptional activator for a superoxide response regulon in Escherichia coli . The activity of SoxR was tested by its ability to enable the transcription of its target gene, soxS, in vitro . The activity of the oxidized form was lost when its {2Fe-2S} clusters were reduced by dithionite under anaerobic conditions, and it was rapidly restored by autooxidation . This result is consistent with the hypothesis that induction of the regulon is effected by the univalent oxidation of the Fe-S centers of SoxR . In vivo, this oxidation may be caused by an alteration of the redox balance of electron chain intermediates that normally maintains soxR in an inactive, reduced state . Oxidized SoxR was about twice as effective as reduced SoxR in protecting the soxS operator from endonucleolytic cleavage . However, this difference could not account for a greater than 50-fold difference in their activities and therefore could not support a model in which oxidation activates SoxR by enabling it to bind to DNA . NADPH, ferredoxin, flavodoxin, or ferredoxin (flavodoxin):NADP+ reductase could not reduce SoxR directly in vitro at a measurable rate . The midpoint potential for SoxR was measured at -283 mV. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10084 - 9 Behavioral responses of Escherichia coli to changes in redox potential; Bespalov VA et al.; Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential . In a spatial redox gradient of benzoquinone/benzoquinol, E . coli cells migrated to form a sharply defined band . Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band . This behavioral response was named redox taxis . Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force . The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector . The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis . A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis . A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein . A similar mechanism has been proposed for aerotaxis . Redox taxis may play an important role in the distribution of bacterial species in natural environments. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10051 - 6 ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation; Wong I et al.; To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S) . Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized . The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16 . Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation . This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer . However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency) . These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA . Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10034 - 9 Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum; Silva AM et al.; Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance . Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted . The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development . We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A . This represents the first reported crystal structure of a protein from P . falciparum . The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme . The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P . falciparum in culture. Biochemistry, 1996 Sep 17, 35(37), 12164 - 74 Phospholipase A2 engineering . Deletion of the C-terminus segment changes substrate specificity and uncouples calcium and substrate binding at the zwitterionic interface; Huang B et al.; It has been suggested {Dijkstra, B . W., Drenth, J., & Kalk, K . H . (1981) Nature 289, 604-606} that the interfacial binding site of phospholipase A2 (PLA2) involves a large number of residues, including a cluster at the N-terminus and another cluster at the C-terminus . The approaches of multiple mutation and deletion were used to test the roles of the C-terminal residues of bovine pancreatic PLA2 overexpressed in Escherichia coli . A double mutant K120A/K121A and a deletion mutant delta 115-123/ C27A were constructed, and structural and functional analyses were performed on both mutants . The double mutant showed little perturbation in the global structure on the basis of proton NMR and X-ray crystallographic analyses . The proton NMR analysis of the deletion mutant suggested that a few residues at the active site, the hydrophobic channel, and the calcium binding loop are perturbed, but the global conformation is not changed . The mutants were then characterized for catalytic and binding properties by use of various kinetic and spectroscopic methods . The double mutant behaved in a manner similar to that of the wild type (WT) PLA2 in every property examined . The deletion mutant was found to show an interesting change of substrate specificity . The kcat,app of the zwitterionic DC8PC micelles but not the anionic DC8PM micelles decreased by a factor of > 100; however, the activity of DC8PC was restored upon addition of 4 M NaCl . The results of fluorescence spectroscopic studies indicate that the deletion mutant behaves in a manner similar to that of WT in the binding to anionic vesicles and to zwitterionic neutral diluent . Thus, the binding affinity of the enzyme to the interface (the E to E* step) should not be the main cause for the change in substrate specificity . The cause lies at least partially in the binding of substrate or inhibitor to the active site of the enzyme at the interface, i.e., the E* to E*L step, as revealed by the results of equilibrium binding studies . The equilibrium dissociation constants of ligands are generally higher for the deletion mutant (relative to WT) at the zwitterionic interface but not at the anionic interface . The cause for the low affinity of an active site-directed ligand to the active site at the zwitterionic interface could be related to the inability of Ca2+ to enhance ligand binding for the deletion mutant . This is in contrast to the WT PLA2 for which Ca2+ binding enhances binding of the substrate to the active site . Overall, the structural and functional perturbations caused by deleting the C-terminal segment are modest, but the changes in substrate specificity and the uncoupling between substrate and calcium binding are interesting and significant. Biochemistry, 1996 Sep 17, 35(37), 12104 - 10 The role of the extrinsic 33 kDa protein in Ca2+ binding in photosystem II; Seidler A et al.; The role of the 33 kDa protein in Ca2+ binding was studied by comparing the EPR properties of photosystem II in the presence and absence of the 33 kDa protein and Ca2+ . When the removal of the 33 kDa protein was carried out in the dark, a normal manganese multiline EPR signal could be observed when the S2 state was generated . In addition, the split S3 signal could not be generated by illumination at 273 K . Exposure of the 33 kDa protein-less photosystem II to room light did not lead to any change in the EPR properties of the S2 state, but the split S3 state signal at around g = 2 could then be generated, indicating that Ca2+ was released from this preparation during the exposure to light . Treatment of photosystem II lacking the 33 kDa protein with EGTA in the light led to a modification of the S2 state characterized by a dark-stable multiline EPR signal . Much lower EGTA concentrations were required in order to obtain this modification in the absence of the 33 kDa protein than was required when the 33 kDa protein was present . This indicates that the manganese cluster was more accessible to chelator binding when the 33 kDa protein was absent . When 33 kDa protein-less photosystem II was treated with EGTA in the dark, no modification of the multiline EPR signal of the S2 state of the manganese cluster occurred, nor was Ca2+ released as monitored by the inability to generate the split S3 signal . These chelator- and Ca(2+)-binding properties occurring in PSII lacking the 33 kDa protein are very similar to those observed previously in NaCl-washed PSII in which the 33 kDa protein is present (reviewed in Boussac & Rutherford, 1994a) . It is concluded that the 33 kDa protein has little or no direct role in binding the Ca2+ ion which is required for oxygen evolution. Biochemistry, 1996 Sep 17, 35(37), 12077 - 85 Alternative splicing determines the binding of platelet-derived growth factor (PDGF-AA) to glycosaminoglycans; Lustig F et al.; We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin . Here, we have expressed the long (rA125) and the short (rA109) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers . Surface plasmon resonance analyses showed that while the dimeric rA125 bound with high affinity to low molecular weight heparin, the rA109, lacking the basic extension, did not . This strongly indicated that high affinity binding is due to the carboxyl-terminal extension . Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism . Thus, dimeric rA125 contains two equivalent binding sites . Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times . This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule . Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement . Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg111 and Lys116, and the polar Thr125 . We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA125 and that heparan sulfate showed the highest affinity. Biochemistry, 1996 Sep 17, 35(37), 12053 - 60 Melibiose permease of Escherichia coli: structural organization of cosubstrate binding sites as deduced from tryptophan fluorescence analyses; Mus-Veteau I et al.; Binding of the coupling ion (Na+ or Li+) and sugars to the purified melibiose permease of Escherichia coli, reconstituted in proteoliposomes, produces selective and cooperative changes of the transporter tryptophan fluorescence . To assess the individual contribution of N- or C-terminal domains of the permease to these substrate-induced fluorescence variations, we replaced the two tryptophans located in its C-terminal half (W299 and W342) by a phenylalanine and compared the signal change in mutants and wild-type permease . None of the mutations significantly impairs transport activity . Persistence of the ion-induced signal quenching in a permease carrying only the six other tryptophans of the N-terminal domain is consistent with a previous suggestion that this domain accommodates the ion-binding site . On the other hand, the sugar-induced fluorescence increase varies from mutant to mutant in a sugar-specific fashion . While alpha-galactosides increase essentially the fluorescence of W299 and W342, beta-galactosides enhance the signal of W299 and of one (or more) of the N-terminal tryptophans but quench that of W342 . Moreover, addition of sugars producers a 10 nm blue shift of both W299 and W342 emission spectra, suggesting reduced accessibility of these residues to solvent following substrate binding . These data suggest that W299 and W342 are at or close to the sugar binding site and that this latter is lined by the C-terminal helices IX and X . Moreover, as sugars with the beta-configuration also enhance the fluorescence of the N-terminal tryptophans, it is suggested that one (or more) helix of the N-terminal half may be also at or near the sugar binding site . This implies close proximity and/or tight functional linkage between some N-terminal helices and helices IX and X of the C-terminal domain of the transporter. Biochemistry, 1996 Sep 17, 35(37), 12046 - 52 Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in escherichia coli; Pyle LE et al.; Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage . Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively . Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central region . While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein . While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers . None of the truncated proapoAI molecules formed oligomers larger than trimers . The shortest form, proapoAI-6-135, only dimerized . Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein . Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI. Biochemistry, 1996 Sep 17, 35(37), 12029 - 37 Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain; Yoo BC et al.; The activity of calmodulin-like domain protein kinase (CDPK) is regulated by the direct binding of Ca2+ . Unmodified soybean CDPK alpha and a chimeric enzyme in which the calmodulin-like domain (CLD) was replaced by VU-1 calmodulin had similar values of Vmax(app) (3.19, 3.46, and 3.60, 3.93 mumol/ min/mg, respectively), and each was activated 30-70-fold by Ca2+ . To determine if activation results from the binding of the CLD to the autoinhibitory (junction) domain of CDPK alpha in a manner analogous to the activation of calmodulin-dependent enzymes by calmodulin, recombinant CLD and truncation mutants of CDPK alpha were expressed in bacteria and highly purified . In blot overlays, biotinylated CLD bound to mutants containing residues 312-328 of the junction domain . In an electrophoretic mobility shift assay CLD bound synthetic peptides containing residues 318-332 in a calcium-dependent manner, providing direct evidence for binding of CLD to a site in the junction domain . Mutants of CDPK alpha from which all or part of the CLD had been deleted were constitutively inactive . Addition of 20 microM CLD to these mutants in the presence, but not the absence, of calcium stimulated their activities, but to various degrees . His6-CDPK alpha (1-328), which contained none of the CLD, was activated only 5-fold, but the activity of His6-CDPK alpha (1-398), which retained nearly half of the CLD in its sequence, was stimulated 64-fold . The latter activity approached that of unmodified CDPK alpha and was half maximal at a CLD concentration of 7 microM . Our results suggest that binding of CLD to the junction domain contributes to, but is not sufficient for activation . Although calmodulin supported full activity of the chimeric enzyme, its addition to His6-CDPK alpha (1-398) resulted in activity that was only 6% of that of the unmodified enzyme and which was half-maximal at 20 microM Arabidopsis calmodulin . These results support the conclusion that simple binding of the calmodulin-like domain to the junction domain is not sufficient for activation. Biochemistry, 1996 Sep 17, 35(37), 11994 - 2004 Domain interactions of the peripheral preprotein Translocase subunit SecA; den Blaauwen T et al.; The homodimeric SecA protein is the peripheral subunit of the preprotein translocase in bacteria . It binds the preprotein and promotes its translocation across the bacterial cytoplasmic membrane by nucleotide modulated coinsertion and deinsertion into the membrane . SecA has two essential nucleotide binding sites (NBS; Mitchell & Oliver, 1993): The high-affinity NBS-I resides in the amino-terminal domain of the protein, and the low-affinity NBS-II is localized at 2/3 of the protein sequence . The nucleotide-bound states of soluble SecA were studied by site directed tryptophan fluorescence spectroscopy, tryptic digestion, differential scanning calorimetry, and dynamic light scattering . A nucleotide-induced conformational change of a carboxy-terminal domain of SecA was revealed by Trp fluorescence spectroscopy . The Trp fluorescence of a single Trp SecA mutant containing Trp775 decreased and increased upon the addition of NBS-I saturating concentrations of ADP or AMP-PNP, respectively . DSC measurements revealed that SecA unfolds as a two domain protein . Binding of ADP to NBS-I increased the interaction between the two domains whereas binding of AMP-PNP did not influence this interaction . When both NBS-I and NBS-II are bound by ADP, SecA seems to have a more compact globular conformation whereas binding of AMP-PNP seems to cause a more extended conformation . It is suggested that the compact ADP-bound conformation resembles the membrane deinserted state of SecA, while the more extended ATP-bound conformation may correspond to the membrane inserted form of SecA. Biochemistry, 1996 Sep 17, 35(37), 11985 - 93 Crystal structure of glycine N-methyltransferase from rat liver; Fu Z et al.; Glycine N-methyltransferase (GNMT) from rat liver is a tetrameric enzyme with 292 amino acid residues in each identical subunit and catalyzes the S-adenosylmethionine (AdoMet) dependent methylation of glycine to form sarcosine . The crystal structure of GNMT complexed with AdoMet and acetate, a competitive inhibitor of glycine, has been determined at 2.2 A resolution . The subunit of GNMT forms a spherical shape with an extended N-terminal region which corks the entrance of active site of the adjacent subunit . The active site is located in the near center of the spherical subunit . As a result, the AdoMet and acetate in the active site are completely surrounded by amino acid residues . Careful examination of the structure reveals several characteristics of GNMT . (1) Although the structure of the AdoMet binding domain of the GNMT is very similar to those of other methyltransferases recently determined by X-ray diffraction method, an additional domain found only in GNMT encloses the active site to form a molecular basket, and consequently the structure of GNMT looks quite different from those of other methyltransferases . (2) This unique molecular structure can explain why GNMT can capture folate and polycyclic aromatic hydrocarbons . (3) The unique N-terminal conformation and the subunit structure can explain why GNMT exhibits positive cooperativity in binding AdoMet . From the structural features of GNMT, we propose that the enzyme might be able to capture yet unidentified molecules in the cytosol and thus participates in various biological processes including detoxification of polycyclic aromatic hydrocarbons . In the active site, acetate binds near the S-CH3 moiety of AdoMet . Simple modeling indicates that the amino group of the substrate glycine can be placed close to the methyl group of AdoMet within 3.0 A and form a hydrogen bond with the carboxyl group of Glu15 of the adjacent subunit . On the basis of the ternary complex structure, the mechanism of the methyl transfer in GNMT has been proposed. Biochemistry, 1996 Sep 17, 35(37), 11967 - 74 A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution; Hara T et al.; The crystal structure of glutathione synthetase from Escherichia coli B complexed with ADP, glutathione, and sulfate has been determined at 2.0 A resolution . Concerning the chemical similarity of sulfate and phosphate, this quaternary complex structure represents a pseudo enzyme-substrate complex in the reverse reaction and consequently allows us to understand the active site architecture of the E . coli glutathione synthetase . Two Mg2+ ions are coordinated with oxygen atoms from the alpha- and beta-phosphate groups of ADP and from the sulfate ion . The flexible loops, invisible in the unliganded or the binary and ternary complex structures, are fixed in the quaternary complex . The larger flexible loop (Ile226-Arg241) includes one turn of a 310-helix that comprises the binding site of the glycine moiety of GSH . The small loop (Gly164-Gly167) is involved in nucleotide binding and acts as a phosphate gripper . The side chains of Arg210 and Arg225 interact with the sulfate ion and the beta-phosphate moiety of ADP . Arg 210 is likely to interact with the carboxylate of the C-terminal gamma-glutamylcysteine in the substrate-binding form of the forward reaction . Other positively charged residues in the active site (Lys125 and Lys160) are involved in nucleotide binding, directing the phosphate groups to the right position for catalysis . Functional aspects of the active site architecture in the substrate-binding form are discussed. Biochemistry, 1996 Sep 17, 35(37), 11951 - 8 Structure of the acid state of Escherichia coli ribonuclease HI; Dabora JM et al.; Under acidic conditions Escherichia coli ribonuclease HI* (RNase H*) adopts a partially folded state with all of the properties of a molten globule . Using amide hydrogen exchange carried out under acid state conditions, followed by quenching and NMR detection on the native state, we have determined the residues that are responsible for the observed structure of the acid state . Although RNase H* is a mixed alpha + beta protein, a helical subdomain (helices A, D, and B) defines the structure of the acid state . This structure correlates with the rare higher energy conformations detected under native conditions and with data for the earliest intermediates populated in the kinetic folding pathway of the protein. Gene, 1996 Sep 16, 173(2), 271 - 4 Production of the beta-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence; Pillai D et al.; The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss) . BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli . Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody . The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction . Expression was evident soon after the addition of the lactose analogue, IPTG . These results demonstrate that E . coli cells can synthesize, process and export betahCG using the LTBss. Gene, 1996 Sep 16, 173(2), 261 - 4 Cloning and sequence analysis of the human salivary peroxidase-encoding cDNA; Kiser C et al.; A cDNA (hSPO) encoding human salivary peroxidase (hSPO) was isolated and its nucleotide (nt) sequence determined . The coding sequence resembles those of other mammalian peroxidases and contains a 3' untranslated region of 538 nt . In a rabbit reticulocyte lysate transcription/trans