Infectious disease and sociodemographic characteristics of foreign immigrants in the Central Penitentiary for Men in Barcelona

A number of thrombin mutants have been constructed to investigate the role of Trp96 and the beta-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the beta-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr(60A)Pro(60B)Pro(60C)Trp(60D) Asp(60E)Lys(60F) of the beta-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp96 --> Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen approximately 3 times more slowly than thrombin, with the two beta-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting approximately 3000- and 1300-fold more slowly, respectively. The specificity constant kcat/Km for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 microM(-1) s(-1), respectively, compared to 10 and 2.5 microM(-1) s(-1) for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant Km increased approximately 6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant kcat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the beta-insertion loop is important for thrombin activity. But the mutation of Trp96 --> Ser can compensate somewhat for the loss of binding at the beta-insertion loop. The deletion of the hydrophobic interaction between Trp96 and Pro(60B)Pro(60C) appears to decrease the stability of the beta-insertion loop, thereby causing a decrease in binding efficiency.     

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.     

Localization of the active site of type II dehydroquinases. Identification of a common arginine-containing motif in the two classes of dehydroquinases

A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the kcat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced Km values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.     

The roles of conserved carboxylate residues in IMP dehydrogenase and identification of a transition state analog

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+; the enzyme is activated by K+. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. In order to identify functionally important residues in IMPDH, including those involved in substrate and K+ binding, we have mutated 11 conserved Asp and Glu residues to Ala in Escherichia coli IMPDH. The values of kcat, Km, and Ki for GMP, XMP, mizoribine 5'-monophosphate (MMP), and beta-methylene-tiazofurin adenine dinucleotide (TAD) were determined. Five of these mutations caused a significant change (>/=10-fold) in one of these parameters. The Asp248 --> Ala mutation caused 100-fold decrease in the value of kcat and a 25-fold increase in the value of Kii for TAD; these observations suggest that Asp248 is in the NAD+ binding site. The Asp338 --> Ala mutation caused a 600-fold decrease in the value of kcat, but only a 5-10-fold increase in the values of Km for IMP and Kis for IMP analogs, suggesting that Asp338 may be involved in acid-base catalysis as well as IMP binding. The remaining three residues, Asp13, Asp50, and Glu469, appear to be involved in K+ activation; these residues may be ligands at one or more K+ binding sites. Interestingly, changes in the values of Ki for MMP correlate with changes in kcat/KmKm of IMPDH, while no such correlation is observed for GMP, XMP, and TAD. This observation indicates that MMP is a transition state analog for the IMPDH reaction.     

Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.     

ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis

The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.     

Site-directed mutagenesis of the active site glutamate in human matrilysin: investigation of its role in catalysis

Glu-198 of human matrilysin is a conserved residue in the matrix metalloproteinases and is considered to play an important role in catalysis by acting as a general base catalyst toward the zinc-bound water molecule, on the basis of mechanistic proposals for other zinc proteases. In the present study, Glu-198 is mutated into Asp, Cys, Gln, and Ala, and the zinc binding properties, kinetic parameters, and pH dependence of each mutant are determined in order to examine the role of Glu-198 in catalysis. The mutations chosen either modify (C and D) or eliminate (A and Q) the general base properties of residue-198. All the mutants bind 2 mol of zinc per mol of enzyme, indicating that Glu-198 is not crucial to the binding of the catalytic zinc to the enzyme. The value of kcat/Km for the E198D mutant is only 4-fold lower than that of wild-type enzyme at the pH optimum of 7.5, while that for the E198C mutant is decreased by 160-fold. The E198Q and E198A enzymes containing the mutations that have eliminated the nucleophilic and acid/base properties of the residue are still active, having lower kcat/Km values of 590- and 1900-fold, respectively. The decrease in activity of all the mutants is essentially due to a decrease in kcat. The kcat/Km values of the mutants as a function of pH display broad bell-shaped curves that are similar to the wild-type enzyme. The acidic pKa value is not greatly affected by the change in the chemical properties of residue-198. The similarity in the pH profiles for the mutant and wild-type enzymes indicates that the ionization of Glu-198 is not responsible for the acidic pKa. Ionization of the zinc-bound water may be responsible for this pKa since the three His ligands and the scaffolding of the matrilysin catalytic zinc site are different from that observed in carboxypeptidase A and would predict a lower pKa for the metal-bound water. If the zinc-bound water is the nucleophile in the reaction, the role of Glu-198 in catalysis may be to stabilize the transition state or act as a general acid catalyst after the rate-determining step.     

Directed evolution of an aspartate aminotransferase with new substrate specificities

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.     

Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues

Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.     

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.     

Hydrolysis of phosphodiesters through transformation of the bacterial phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.     

Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (-1.86 to 1 degree C) and three South American (4 to 10 degree C) notothenioid teleosts. Higher Michaelis-Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs.     

Effect of an amino acid insertion into the omega loop region of a class C beta-lactamase on its substrate specificity

The extended-substrate specificity of Enterobacter cloacae GC1 beta-lactamase is entirely due to a three amino acid insertion after position 207. To clarify the reason for the extended-substrate specificity, Ala, Ala-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala were inserted after position 207 on the basis of the class C beta-lactamase from E. cloacae P99, respectively. The kcat and Km values of all the mutant enzymes for cephalothin, benzylpenicillin and ampicillin were almost the same as those of the wild-type enzyme, except for those of P99-210-4A which were decreased 4-15-fold. On the other hand, the kcat and Km values for oxyimino beta-lactams such as cefuroxime, ceftazidime, and aztreonam increased with increasing numbers of inserted alanines. The kcat values of the mutant enzymes for cefroxime increased 140-7400-fold compared with that of the wild-type. The Km values also increased with almost the same magnitude, resulting in about the same kcat/Km values as that of the wild-type. On progressive inhibition analysis of aztreonam of the mutant enzymes, two kinds of inactive acyl-enzyme with distinct stabilities were observed, and the proportion of the less stable inactive enzyme increased with increasing numbers of inserted alanines. This suggests that the extension of the substrate specificity is due to instability of the acyl-intermediate caused by an increased deacylation rate in the reaction process.     

Factor VIIa's first epidermal growth factor-like domain's role in catalytic activity

Factor VIIa-tissue factor complex formation initiates the extrinsic blood coagulation pathway. We investigated factor VIIa's first epidermal growth factor-like (egf1) domain's role in the catalytic activity increase caused when factor VIIa binds tissue factor. Starting with a factor VIIa with factor IX's egf1 domain (factor VII(IXegf1)a), we made 4 proteins with egf1 residues changed to those in factor VIIa, including E51A, D64Q, FG74-75PA, and K79R. We measured each enzyme's affinity for tissue factor and determined the enzymes' kinetic constants with and without tissue factor. The Kd for factor VII(IXegf1)a binding to tissue factor was 60-200-fold higher than that of factor VIIa depending on the assay employed. Only factor VII(IXegf1)a with the K79R (K79Ra) mutation, among all the mutants, had an effect on binding with a Kd 3-8-fold lower than that of factor VII(IXegf1)a. In kinetic analyses with a small peptide substrate, in the absence of tissue factor, factor VIIa, factor VII(IXegf1)a, and K79Ra had similar kcat's and Km's. With tissue factor, due to a kcat decrease, factor VII(IXegf1)a's catalytic efficiency (kcat/Km) was 2-fold lower than factor VIIa's. K79Ra's catalytic efficiency was intermediate between those of factor VIIa and factor VII(IXegf1)a. With factor X as substrate, in the absence of tissue factor, K79Ra and factor VII(IXegf1)a had catalytic efficiencies 1.5-fold and 2-fold lower than that of factor VIIa. In contrast, with tissue factor and with factor X as substrate, due to higher Km's, factor VII(IXegf1)a and K79Ra had only 9% and 33% of factor VIIa's catalytic efficiency. Our results suggest the egf1 domain's role in tissue factor binding involves critical alignment of tissue factor with factor VIIa's catalytic domain. Proper alignment in turn promotes optimal catalytic activities.     

Coulombic forces in protein-RNA interactions: binding and cleavage by ribonuclease A and variants at Lys7, Arg10, and Lys66

The interactions between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extend beyond the scissile P-O5' bond. Enzymic subsites interact with the bases and phosphoryl groups of the bound substrate. Those residues interacting with the phosphoryl group comprise the P0, P1, and P2 subsites, with the scissile bond residing in the P1 subsite. Here, the function of the P0 and P2 subsites of RNase A is characterized in detail. Lys66 (P0 subsite) and Lys7 and Arg10 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) [poly(C)] and for their abilities to bind to single-stranded DNA, a substrate analogue. The values of kcat and Km for poly(C) cleavage were affected by altering the P0 and P2 subsites. The kcat/Km values for poly(C) cleavage by the K66A, K7A/R10A, and K7A/R10A/K66A variants were 3-fold, 60-fold, and 300-fold lower, respectively, than that of wild-type RNase A. These values indicate that the P0 and P2 subsites contribute 0.70 and 2.46 kcal/mol, respectively, to transition-state binding. Binding experiments indicate that the P0 and P2 subsites contribute 0.92 and 1.21 kcal/mol, respectively, to ground-state binding. Thus, the P0 subsite makes a uniform contribution toward binding the ground state and the transition state, whereas the P2 subsite differentiates, binding more tightly to the transition state than to the ground state. In addition, nucleic acid binding to wild-type RNase A is strongly dependent on NaCl concentration, but this dependence is diminished upon alteration of the P0 or P2 subsite. The logarithm of Kd is a linear function of the logarithm of [Na+] over the range 0.018 M 相似文献   

Dissecting the contributions of a specific side-chain interaction to folding and catalysis of Bacillus stearothermophilus lactate dehydrogenase

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].     

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.     

Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.