Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.     

Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase

The mammalian mitochondrial NADP-dependent isocitrate dehydrogenase is a citric acid cycle enzyme and an important contributor to cellular defense against oxidative stress. The Mn(2+)-isocitrate complex of the porcine enzyme was recently crystallized; its structure indicates that Ser(95), Asn(97), and Thr(78) are within hydrogen-bonding distance of the gamma-carboxylate of enzyme-bound isocitrate. We used site-directed mutagenesis to replace each of these residues by Ala and Asp. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. All the enzymes retain their native dimeric structures and secondary structures as monitored by native gel electrophoresis and circular dichroism, respectively. V(max) of the three alanine mutants is decreased to 24%-38% that of wild-type enzyme, with further decreases in the aspartate mutants. For T78A and S95A mutants, the major changes are the 10- to 100-fold increase in the K(m) values for isocitrate and Mn(2+). The results suggest that Thr(78) and Ser(95) function to strengthen the enzyme's affinity for Mn(2+)-isocitrate by hydrogen bonding to the gamma-carboxylate of isocitrate. For the Asn(97) mutants, the K(m) values are much less affected. The major change in the N97A mutant is the increase in pK(a) of the ionizable metal-liganded hydroxyl of enzyme-bound isocitrate from 5.23 in wild type to 6.23 in the mutant enzyme. The hydrogen bond between Asn(97) and the gamma-carboxylate of isocitrate may position the substrate to promote a favorable lowering of the pK of the enzyme-isocitrate complex. Thus, Thr(78), Ser(95), and Asn(97) perform important but distinguishable roles in catalysis by porcine NADP-specific isocitrate dehydrogenase.     

Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149

The steady-state kinetic parameters for epimerization of UDP-galactose by UDP-galactose 4-epimerase from Escherichia coli (GalE), Y149F-GalE, and S124A-GalE have been measured as a function of pH. The deuterium kinetic isotope effects for epimerization of UDP-galactose-C-d(7) by these enzymes have also been measured. The results show that the activity of wild-type GalE is pH-independent in the pH range of 5.5-9.3, and there is no significant deuterium kinetic isotope effect in the reaction of UDP-galactose-C-d(7). It is concluded that the rate-limiting step for epimerization by wild-type GalE is not hydride transfer and must be either a diffusional process or a conformational change. Epimerization of UDP-galactose-C-d(7) by Y149F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on k(cat) of 2.2 +/- 0.4 at pH 6.2 and 1.1 +/- 0.5 at pH 8.3. Moreover, the plot of log k(cat)/K(m) breaks downward on the acid side with a fitted value of 7.1 for the pK(a). It is concluded that the break in the pH-rate profile arises from a change in the rate-limiting step from hydride transfer at low pH to a conformational change at high pH. Epimerization of UDP-galactose-C-d(7) by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on k(cat) of 2.0 +/- 0.2 between pH 6 and 9. Both plots of log k(cat) and log k(cat)/K(m) display pH dependence. The plot of log k(cat) versus pH breaks downward with a pK(a) of 6.35 +/- 0.10. The plot of log k(cat)/K(m) versus pH is bell-shaped, with fitted pK(a) values of 6.76 +/- 0.09 and 9.32 +/- 0.21. It is concluded that hydride transfer is rate-limiting, and the pK(a) of 6.7 for free S124A-GalE is assigned to Tyr 149, which displays the same value of pK(a) when measured spectrophotometrically in this variant. Acid-base catalysis by Y149F-GalE is attributed to Ser 124, which is postulated to rescue catalysis of proton transfer in the absence of Tyr 149. The kinetic pK(a) of 7.1 for free Y149F-GalE is lower than that expected for Ser 124, as proven by the pH-dependent kinetic isotope effect. Epimerization by the doubly mutated Y149F/S124A-GalE proceeds at a k(cat) that is lower by a factor of 10(7) than that of wild-type GalE. This low rate is attributed to the synergistic actions of Tyr 149 and Ser 124 in wild-type GalE and to the absence of any internal catalysis of hydride transfer in the doubly mutated enzyme.     

Site-directed mutants of staphylococcal nuclease. Detection and localization by 1H NMR spectroscopy of conformational changes accompanying substitutions for glutamic acid-43

The high-resolution X-ray crystal structure of staphylococcal nuclease suggests that the gamma-carboxylate group of Glu-43 is directly involved in catalysis as a general base that facilitates the attack of water on the substrate phosphodiester. We have used primer-directed, site-specific mutagenesis to generate aspartate, glutamine, asparagine, alanine, and serine substitutions for this residue. The Vmax/Km for the aspartate mutant is reduced 1400-fold and the values for the charge-neutral mutations are reduced 5000-fold relative to the wild-type enzyme. Although these reductions in catalytic efficiency might appear useful in quantitatively estimating the importance of general basic catalysis in the reaction catalyzed by the wild-type enzyme, the thermal stabilities and 1H NMR spectral properties of the mutants suggest that such interpretations are ambiguous. All five mutants have higher melting temperatures for thermal denaturation than the wild-type enzyme, suggesting that the mutants have enhanced thermal stabilities relative to the wild-type enzyme. Chemical shift changes relative to the wild type are observed in both the aromatic and upfield-shifted methyl group regions of the 1H NMR spectra of the aspartate and serine mutants, suggesting the presence of conformational differences between the wild-type and mutant enzymes. That these conformational differences may be large enough to be mechanistically relevant is suggested by comparisons of the magnitudes of nuclear Overhauser effect (NOE) correlations between the aromatic and upfield-shifted methyl group regions observed via two-dimensional nuclear Overhauser effect correlation spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase

Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme. Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively. In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme. The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis. This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085], does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates. This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase.     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the ligand-promoted global conformational change in aspartate transcarbamoylase. Evidence for a two-state transition from boundary spreading in sedimentation velocity experiments

A global conformational change in the regulatory enzyme aspartate transcarbamoylase of Escherichia coli was demonstrated 20 years ago by the 3.5% decrease in the sedimentation coefficient of the enzyme upon its interaction with carbamoyl phosphate and saturating amounts of the aspartate analog succinate. This "swelling" of aspartate transcarbamoylase attributable to the T----R allosteric transition was observed also in subsequent studies when the enzyme was completely saturated with the bisubstrate analog N-(phosphonacetyl)-L-aspartate. In neither of these studies was a direct attempt made by an analysis of boundary spreading (expressed as an apparent diffusion coefficient) on partially liganded enzyme to determine whether the solution contained only T and R-state molecules, as expected for a concerted transition, or a mixture of more than two distinct conformational states. The sensitivity of boundary spreading measurements was tested with a known mixture of fully liganded wild-type enzyme (R-state) and an inactive T-state mutant that did not bind either succinate or the bisubstrate ligand. This experiment yielded broad boundaries with an apparent diffusion coefficient about 10% greater than that of T-state enzyme, due to the differential sedimentation of the two independent species. Identical boundary spreading was obtained theoretically by simulating an equimolar mixture of T and R-state aspartate transcarbamoylase. These results proved that the boundary spreading measurement was sensitive to the presence of heterogeneity. Analogous experiments with only wild-type enzyme in the presence of sub-stoichiometric amounts of the tightly bound bisubstrate ligand sufficient to promote a 1.8% decrease in sedimentation coefficient also exhibited broader boundaries, corresponding to a 10% increase in the apparent diffusion coefficient relative to the unliganded enzyme. In contrast, such broad boundaries were not observed in experiments when the weakly bound succinate was present in quantities sufficient to cause the same 1.8% decrease in sedimentation coefficient. The differences in boundary spreading observed with the two active-site ligands were accounted for by the affinities of the respective ligands for the enzyme and the transport theory of a ligand-promoted isomerization of the protein. In the presence of sub-stoichiometric levels of the tight-binding bisubstrate ligand, the dynamic equilibrium between the T and the R-state is essentially uncoupled and the species sediment at slightly different rates to give broad boundaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.     

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.     

3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism

SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be critical for catalysis by mutating to Ala, while the Cys145 to Ser mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pK(a)'s for His41 and Cys145 in the wild-type enzyme were estimated to be 6.38 and 8.34, while the pK(a)'s for His41 and Ser145 in the C145S mutant were estimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D(2)O for general base catalysis, that is, reacts slower in D(2)O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pK(a) values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.     

A catalytic triad is responsible for acid-base chemistry in the Ascaris suum NAD-malic enzyme

The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K(malate) for the K199R mutant enzyme reveals a pK of 5.3 for an enzymatic group required to be unprotonated for activity and a second pK of 6.3 that leads to a 10-fold loss in activity above the pK of 6.3 to a new constant value up to pH 10. The V profile for K199R is pH independent from pH 5.5 to pH 10 and decreases below a pK of 4.9. Tyrosine 126 is positioned to act as the general acid that donates a proton to the enolpyruvate intermediate to form pyruvate. The pH dependence of V/K(malate) for the Y126F mutant is qualitatively similar to K199R, with a requirement for a group to be unprotonated for activity with a pK of 5.6 and a partial activity loss of about 3-fold above a pK of 6.7 to a new constant value. The Y126F mutant enzyme is about 60000-fold less active than the wild-type enzyme. In contrast to K199R, the V rate profile for Y126F also shows a partial activity loss above pH 6.6. The wild-type pH profiles were reinvestigated in light of the discovery of the partial activity change for the mutant enzymes. The wild-type V/K(malate) pH-rate profile exhibits the requirement for a group to be unprotonated for catalysis with a pK of 5.6 and also shows the partial activity loss above a pK of 6.4. The wild-type V pH-rate profile decreases below a pK of 5.2 and is pH independent from pH 5.5 to pH 10. Aspartate 294 is within hydrogen-bonding distance to K199 in the open and closed forms of malic enzyme. D294A is about 13000-fold less active than the wild-type enzyme, and the pH-rate profile for V/K(malate) indicates the mutant is only active above pH 9. The data suggest that the pK present at about pH 5.6 in all of the pH profiles represents D294, and during catalysis D294 accepts a proton from K199 to allow K199 to act as a general base in the reaction. The pK for the general acid in the reaction is not observed, consistent with rapid tautomerization of enolpyruvate. No other ionizable group in the active site is likely responsible for the partial activity change observed in the pH profiles, and thus the group responsible is probably remote from the active site and the effect on activity is transmitted through the protein by a conformational change.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

Crystallographic snapshot of a productive glycosylasparaginase-substrate complex

Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.     

Cocrystallization of a mutant aspartate aminotransferase with a C5-dicarboxylic substrate analog: structural comparison with the enzyme-C4-dicarboxylic analog complex

A mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB17), previously created by directed evolution, shows increased activity for beta-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild-type enzyme while maintaining the enhanced activity of ATB17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme.     

The role of an active site histidine in the catalytic mechanism of aspartate transcarbamoylase

Although the allosteric enzyme aspartate transcarbamoylase from Escherichia coli has been the focus of numerous physical and enzymological studies for over 2 decades, the catalytic mechanism is still poorly understood. There has been much speculation regarding the role of a conserved histidine residue at position 134 which recent crystallographic studies have implicated in the catalytic mechanism as a general acid or as a general base. We have used a combination of site-directed mutagenesis, 13C-isotope incorporation, and high field NMR to probe the role of His134 in the catalytic mechanism of aspartate transcarbamoylase. By comparing the wild-type catalytic trimer with that from a partially active mutant in which His134 is replaced by alanine, we have assigned the 13C resonance for His134 in the wild-type enzyme. This residue is shown to have a pK less than 6, indicating that the imidazole ring is unprotonated at pH values optimal for enzymatic activity (pH 8.0). This result eliminates the possibility of His134 participating as a general acid in the carbamoyl transfer mechanism. Since the crystallographic studies indicate that His134 is close enough to hydrogen-bond to the carbonyl of the liganded bisubstrate analog N-(phosphonacetyl)-L-aspartate, the imidazole ring would be oriented as to make it unlikely that the N1 lone pair of electrons could participate in general base catalysis. Moreover, if His134 is implicated in base catalysis, we would have expected a much greater loss of activity upon its replacement by alanine. Perhaps the role of His134 is merely to help position the carbonyl group of carbamoyl phosphate for nucleophilic attack by the alpha-amino group of aspartate.     

Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase

Thr(93), Ser(94), Thr(140), and Ser(306) are conserved in all adenylosuccinate lyases (ASL) and are close to other amino acids previously identified by mutagenesis as being in the active site. To test their involvement in the enzyme's function, each of these amino acids was replaced by alanine. All the mutants exhibit circular dichroism spectra which are similar to that of wild-type enzyme, indicating there is no appreciable change in secondary structure. T93A exhibits 0.5% of the V(max) of wild-type ASL with a 10-fold increase in K(m) for adenylosuccinate. S94A has 65% of the V(max) of wild-type ASL with little change in K(m). T140A exhibits 0.03% of the activity of wild-type enzyme with an 11-fold increase in K(m). S306A has 0.4% of the V(max) of wild-type ASL with a sevenfold increase in K(m). Measurements of the pH-V(max) profile reveal a pK(2) value for S94A of 7.83 and S306A of 7.65, in contrast to 8.24 for the wild-type enzyme and 8.42 for T93A. Thr(93) may orient adenylosuccinate optimally for catalysis, while Ser(94) stabilizes protonated His(89), a determinant of pK(2). Thr(140) may, through hydrogen bonding, interact with Asn(270), an amino acid essential for catalysis. Ser(306) may be involved in a hydrogen bond network that ultimately stabilizes protonated His(68), which is probably the general acid in the reaction of enzyme with substrate. The results of this paper demonstrate the importance in the catalytic function of ASL of hydrogen bonds and hydrogen bonding networks involving serine and threonine.     

A single amino acid substitution in the active site of Escherichia coli aspartate transcarbamoylase prevents the allosteric transition

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side-chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved, small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7 angstroms resolution. Time-resolved, small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side-chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side-chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate-binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low-activity low-affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate-binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase.