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.     

Isolation and characterization of the heat-responsive genes in Escherichia coli

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.     

Intramolecular proton transfer from multiple sites in catalysis by murine carbonic anhydrase V

The hydration of CO2 catalyzed by carbonic anhydrase requires proton transfer from the zinc-bound water at the active site to solution for each cycle of catalysis. In the most efficient of the mammalian carbonic anhydrases, isozyme II, this transfer is facilitated by a proton shuttle residue, His 64. Murine carbonic anhydrase V (mCA V) has a sterically constrained tyrosine at the analogous position; it is not an effective proton shuttle, yet catalysis by this isozyme still achieves a maximal turnover in CO2 hydration of 3 x 10(5) s-1 at pH > 9. We have investigated the source of proton transfer in a truncated form of mCA V and identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contribute to proton transfer. Intramolecular proton-transfer rates between these shuttle groups and the zinc-bound water were estimated as the rate-determining step in kcat for hydration of CO2 measured by stopped-flow spectrophotometry and in the exchange of 18O between CO2 and water measured by mass spectrometry. Comparison of kcat in catalysis by Lys 91 and Tyr 131 and the corresponding double mutant showed a strong antagonistic interaction between these sites, suggesting a cooperative behavior in facilitating the proton-transfer step of catalysis. Replacing four potential proton shuttle residues produced a multiple mutant that had 10% of the catalytic turnover kcat of the wild type, suggesting that the main proton shuttles have been accounted for in mCA V. These replacements caused relatively small changes in kcat/Km for hydration, which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers; these measurements serve as a control indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.     

Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.     

Catalytic contribution of phenylalanine-101 of 3-oxo-Delta 5-steroid isomerase

3-Oxo-delta 5-steroid isomerase (KSI, EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated Delta4-isomers through the formation of an intermediate dienolate ion. It has previously been found in our laboratory that the aromatic ring of Phe-101 is important for catalysis. The present work extends these studies. Two double-mutant KSIs (D38E/F101L and D38E/F101A) were prepared to compare the free energy profiles for the reactions catalyzed by these mutants and by D38E. Both double-mutant KSIs show reduced values of kcat at pH 7 compared to D38E ( approximately 25-fold for D38E/F101L and approximately 200-fold for D38E/F101A), similar to the reduced values for F101L and F101A relative to KSI ( approximately 30-fold for F101L and approximately 270-fold for F101A). Free energy profiles for the reactions catalyzed by D38E/F101L and D38E/F101A indicate that the bound transition state(s) and bound intermediate are destabilized when the large aromatic residue Phe-101 in D38E KSI is replaced by the smaller residues Leu or Ala. The pH-rate profiles for D38E, D38E/F101L, and D38E/F101A in the pH range 3.9-8.7 show that the pKa of the catalytic base (Glu-38) is perturbed. In addition, these mutants have significant catalytic activity in the low-pH region.     

Kinetic and spectroscopic investigations of wild-type and mutant forms of apple 1-aminocyclopropane-1-carboxylate synthase

Two catalytically inactive mutant forms of 1-aminocyclopropane-1-carboxylate (ACC) synthase, Y85A and K273A, were mixed in low concentrations of guanidine hydrochloride (GdnHCl). About 15% of the wild-type activity was recovered (theoretical 25% for a binomial distribution), proving that the functional unit of the enzyme is a dimer, or theoretically, a higher order oligomer. The enzyme catalyzes the conversion of S-adenosyl-L-methionine (SAM) to ACC. The value of kcat/KM is 1.2 x 10(6) M-1 s-1 at pH 8.3. Viscosity variation experiments with glycerol and sucrose as viscosogenic reagents showed that this reaction is nearly 100% diffusion controlled. The sensitivity to viscosity for the corresponding reaction of the less reactive Y233F mutant is much reduced, thus the latter reaction serves as a control for that of the wild-type enzyme. The kcat/KM vs pH profile for wild-type enzyme exhibits pKa values of 7.5 and 8.9. The former is assigned to the pKa of the alpha-amino group of SAM, while the latter corresponds to the independently determined spectrophotometric pKa of the internal aldimine. The kcat vs pH profile exhibits similar pKas, which means that the above pKa values are not perturbed in the Michaelis complex. The phenolic hydroxyl group of Tyr233 forms a hydrogen bond to the 3'-O- of PLP. The spectral and kinetic pKa (kcat/KM) values of the Y233F mutant are not identical (spectral 10.2, kinetic 8.7). A model that accounts quantitatively for these data posits two parallel pathways to the external aldimine for this mutant, the minor one has the alpha-amino group free base form of SAM reacting with the protonated imine form of the enzyme with kcat/KM approximately 6.0 x 10(3) M-1 s-1, while the major pathway involves reaction of the aldehyde form of PLP with SAM with kcat/KM approximately 7.0 x 10(5) M-1 s-1. The spectral pKa is defined only by the less reactive species.     

The catalytic properties of murine carbonic anhydrase VII

Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.     

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.     

Spatially varying equilibria of mechanical models: application to dermal wound contraction

We have determined structures of binary and ternary complexes of five Asn229 variants of thymidylate synthase (TS) and related their structures to the kinetic constants measured previously. Asn229 forms two hydrogen bonds to the pyrimidine ring of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). These hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP, and in ternary complexes with dUMP and the TS cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. In N229 mutants, where these hydrogen bonds cannot be made, dUMP binds in a misoriented or more disordered fashion. Most N229 mutants exhibit no activity for the dehalogenation of 5-bromo-dUMP, which requires correct orientation of dUMP against Cys198. Since bound dUMP forms the binding surface against which the pterin ring of cofactor binds, misorientation of dUMP results in higher Km values for cofactor. At the same time, binding of the cofactor aids in ordering and positioning dUMP for catalysis. Hydrophobic mutants, such as N229I, favor an arrangement of solvent molecules and side-chains around the ligands similar to that in a proposed transition state for ternary complex formation in wild-type TS, and kcat values are similar to the wild-type value. Smaller, more hydrophilic mutants favor arrangements of the solvent and side-chains surrounding the ligands that do not resemble the proposed transition state. These changes correspond to decreases in kcat of up to 2000-fold, with only modest increases in Km or Kd. These results are consistent with the proposal that the hydrogen-bonding network between water, dUMP and side-chains in the active-site cavity contributes to catalysis in TS. Asn229 has the unique ability to maintain this critical network, without sterically interfering with dUMP binding.     

Purification of active calpain by affinity chromatography on an immobilized peptide inhibitor

The 19F-NMR resonance of 5-[19F]fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine. Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M. Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8). 5-[19F]Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M). 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state. These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state.     

Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.     

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].     

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)     

Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.     

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

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.     

The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate

S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif. Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.     

Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a

Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases. Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties. With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes. There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH. Y6F mutations have no effect on the pKa for these enzymes. Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group. Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation. All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher. The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases. Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting.     

Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor

5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.     

Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis

Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.