Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies

The pH dependence of kinetic parameters and inhibitor dissociation constants for the adenosine cyclic 3',5'-monophosphate dependent protein kinase reaction has been determined. Data are consistent with a mechanism in which reactants selectively bind to enzyme with the catalytic base unprotonated and an enzyme group required protonated for peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) binding. Binding of the peptide apparently locks both of the above enzyme residues in their correct protonation state. MgATP preferentially binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/KMgATP are pH independent. The V/K for Ser-peptide is bell-shaped with pK values of 6.2 and 8.5 estimated. The pH dependence of 1/Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/KSer-peptide, while the Ki for MgAMP-PCP increases from a constant value of 650 microM above pH 8 to a constant value of 4 mM below pH 5.5. The Ki for uncomplexed Mg2+ obtained from the Mg2+ dependence of V and V/KMgATP is apparently pH independent.     

Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli

Site-directed mutagenesis was used to investigate the importance of Lys178, Arg286, and Arg294 in the binding of prephenate to the bifunctional enzyme chorismate mutase-prephenate dehydrogenase. From comparison of the kinetic parameters of wild-type enzyme and selected mutants, we conclude that only Arg294 interacts specifically with prephenate. The R294Q substitution reduces the enzyme's affinity for prephenate without affecting V/Et of the dehydrogenase reaction or the kinetic parameters of the mutase reaction. Arg294 likely interacts with the ring carboxylate at C-1 of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxyl group were similar for wild-type and R294Q enzymes. The pH dependencies of log (V/KprephenateEt) and of pKi for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be protonated for binding prephenate to the enzyme. None of the three conserved residues is this group since its titration is observed in the V/KprephenateEt profiles for the mutants K178Q, R286A, and R294Q. This group is also seen in the pH-rate profiles of the binding of two substrate analogues, hydroxyphenyllactate and deoxoprephenate. Their only common structural feature at C-1 is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate. Gdn-HCl-induced denaturation was conducted on wild-type and selected mutant proteins. Unfolding of the wild-type enzyme proceeds through a partially unfolded dimer which dissociates into unfolded monomers. The order of stability is wild-type = R294Q > K178Q > R286A > K178R. The least unstable mutants have reduced mutase and dehydrogenase activities.     

Chemical mechanism of saccharopine dehydrogenase (NAD+, L-lysine-forming) as deduced from initial rate pH studies

The variation of kinetic parameters with pH has been determined so as to gain insight into the chemical mechanism of the saccharopine dehydrogenase (NAD+,L-lysine-forming)-catalyzed reaction. In the direction of reductive condensation of lysine and alpha-ketoglutarate (reverse reaction), the V/K profile for lysine shows a group with a pK of 6.3 must be unprotonated and a group with a pK of 8.0 must be protonated for activity. Similar pK's are obtained in the pKi profile for ornithine, which acts as a linear competitive inhibitor with respect to lysine. Temperature and solvent perturbation studies show that these groups are probably histidines. The V/K profile for alpha-ketoglutarate reveals a single group with pK = 8.4 (probably lysine) that must be protonated. It is proposed that one of the histidines is involved in the binding of the epsilon-amino group of the substrate lysine and the positively charged lysine residue hydrogen bonds to the carbonyl oxygen of alpha-ketoglutarate. In the direction of saccharopine cleavage, the V/K profile for saccharopine shows that two groups with pK values of 6.0 and 7.1, possibly a histidine and lysine, must be unprotonated for its reaction with the enzyme X NAD+ complex. The log V-pH plots for the forward and reverse reactions both show sigmoidal curves. At low pH, the activity is lower for the forward reaction, and is higher for the reverse reaction. The ionization of a single group appears to be responsible for the change in activity. A tentative scheme for the chemical reaction is presented.     

Chemical mechanism of beta-xylosidase from Trichoderma reesei QM 9414: pH-dependence of kinetic parameters.

The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM 9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a group on the enzyme with a pK value of 3.20 must be unprotonated and a group with a pK value of 5.20 must be protonated for activity and both are involved in catalysis. Solvent-perturbation studies indicated that these groups are neutral acid type. Temperature dependence of kinetic parameters suggested the stickiness of the substrate at lower temperatures than the optimum and the calculated ionization enthalpies pointed to carboxyl groups as responsible for both pKs. Chemical modification with triethyloxonium tetrafluoroborate and protection with the substrate studies demonstrated essential carboxyl groups on the enzyme. Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK value of 3.45 must be protonated for binding and it has been assigned to the carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.     

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)     

Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

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.     

Chemical mechanism of Haemophilus influenzae diaminopimelate epimerase

Seven unique enzymatic steps lead to the biosynthesis of L-lysine from L-aspartate semialdehyde and pyruvate in bacteria. The immediate precursor to L-lysine is D,L-diaminopimelate, a diamino acid which is incorporated into the pentapeptide of the Gram-negative peptidoglycan moiety. D,L-Diaminopimelate is generated from the corresponding L,L-isomer by the dapF-encoded epimerase. Diaminopimelate epimerase is a representative of the pyridoxal phosphate-independent amino acid racemases, for which substantial evidence exists supporting the role of two cysteine residues as the catalytic acid and base. The pH dependencies of the maximum velocities in the L,L --> D,L and D,L --> L,L direction depend on a single catalytic group exhibiting pK values of 7.0 and 6.1, respectively, which must be unprotonated for activity. The pH dependencies of the V/K values in both directions depend on the ionization of two groups, one exhibiting a pK value of 6.7 which must be unprotonated and one exhibiting a pK value of 8.5 which must be protonated. Primary kinetic isotope effects on V and V/K are unequal, with D(V/K) being larger than DV in both the forward and reverse directions. Solvent kinetic isotope effects in both directions are inverse on V/K, but normal on V. Both of these isotopic observations support a model in which proton isomerization after catalysis and substrate dissociation is kinetically significant. A single solvent "overshoot" is observed when L, L-diaminopimelate is incubated with enzyme in D2O; however, an unprecedented double overshoot is observed when D,L-diaminopimelate is incubated with enzyme in D2O. A model has been developed which allows these two overshoots to be simulated. A chemical mechanism is proposed invoking the function of two cysteine residues, Cys73 and Cys217, observed in the recently determined three-dimensional structure of this enzyme [Cirilli, M., et al. (1998) Biochemistry 37, 16452-16458], as the acid and base in the mechanism.     

Catalytic mechanism of the tryptophan synthase alpha(2)beta(2) complex. Effects of pH, isotopic substitution, and allosteric ligands.

The mechanism of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK(a)1 = 6.5, pK(a)2 = 7.3, and pK(a)3 = 8.2-9. A significant primary isotope effect (approximately 3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the alpha-proton (betaLys-87) is protonated at low pH, slowing the abstraction of the alpha-proton and making this step at least partially rate-limiting. pK(a)2 is assigned to betaLys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK(i) values of 7.3. The residue with this pK(a) (betaLys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK(a) values support the assignment of pK(a)2 to betaLys-87 and suggest that the ionizing residue with pK(a)1 could be a carboxylate, possibly betaAsp-305, and that the residue associated with a conformational change at pK(a)3 may be betaLys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.     

Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/KNADPH profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the Ki profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/KNADPH, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123] to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. At neutral pH, the proportion of enzyme present as a protonated ternary enzyme-substrate complex is sufficient to keep catalysis faster than product release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid-base catalysis in the argininosuccinate lyase reaction

The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward reaction the Vmax profile showed one group that must be unprotonated for activity over the pH range 5-10. The V/K profile for argininosuccinate showed one group that must be unprotonated and two groups that must be protonated for activity. The Vmax profile for the reverse reaction showed only one group that must be protonated for activity. These results support the proposal that catalysis is facilitated in the forward reaction by a general base that abstracts a proton from C-3 of argininosuccinate and a general acid that donates a proton to the guanidinium nitrogen during carbon-nitrogen bond cleavage. The enzyme is completely inactivated by diethyl pyrocarbonate or a water-soluble carbodiimide at pH 6. These experiments suggest that a histidine and a carboxyl group are at or near the active site and are essential for catalytic activity. The observed shifts of the pH profiles of the forward reaction with temperature and organic solvent (25% dioxane) were also consistent with a histidine and carboxylate group.     

Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism

The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.     

Catalytic mechanism of the dihydrofolate reductase reaction as determined by pH studies

The variation with pH of the kinetic parameters of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been determined with the aim of elucidating the chemical mechanism of the reaction. The (V/K)DHF and V profiles indicated that protonation enhances the observed rate of interaction of dihydrofolate (DHF) with the enzyme-NADPH complex as well as the maximum velocity of the reaction. The pKa value of 8.09 observed in the (V/K)DHF profile is similar to that of 7.9 observed in the Ki profile for 2,4-diamino-6,7-dimethylpteridine while the pKa value of the V profile is displaced to 8.4. From the magnitude of the pH-independent value for (V/K)DHF, it is concluded that unprotonated dihydrofolate must react, at neutral pH, with the protonated form of the enzyme. The D(V/K)DHF value is independent of pH and equal to unity whereas the DV value varies as a wave function of pH with limiting values of 1.5 and 1.0 at low and high pH, respectively. It is proposed that dihydrofolate reacts with the unprotonated enzyme-NADPH complex to form a dead-end complex and with the protonated form of the same complex to form a productive complex. Further, it is considered that the protonated carboxyl of Asp-27 at the active site of the enzyme is responsible for the protonation of the N-5 nitrogen of dihydrofolate and that this protonation precedes and facilitates hydride transfer.     

pH dependence of kinetic parameters for oxalacetate decarboxylation and pyruvate reduction reactions catalyzed by malic enzyme

Both chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzyme catalyze the metal-dependent decarboxylation of oxalacetate. Both enzymes catalyze the reaction either in the presence or in the absence of dinucleotide. The presence of dinucleotide increases the affinity of oxalacetate for the chicken liver NADP-malic enzyme, but this information could not be obtained in the case of A. suum NAD-malic enzyme because of the low affinity of free enzyme for NAD. The kinetic mechanism for oxalacetate decarboxylation by the chicken liver NADP-malic enzyme is equilibrium ordered at pH values below 5.0 with NADP adding to enzyme first. The Ki for NADP increases by a factor of 10 per pH unit below pH 5.0. An enzyme residue is required protonated for oxalacetate decarboxylation (by both enzymes) and pyruvate reduction (by the NAD-malic enzyme), but the beta-carboxyl of oxalacetate must be unprotonated for reaction (by both enzymes). The pK of the enzyme residue of the chicken liver NADP-malic enzyme decreases from a value of 6.4 in the absence of NADP to about 5.5 with Mg2+ and 4.8 with Mn2+ in the presence of NADP. The pK value of the enzyme residue required protonated for either oxalacetate decarboxylation or pyruvate reduction for the A. suum NAD-malic enzyme is about 5.5-6.0. Although oxalacetate binds equally well to protonated and unprotonated forms of the NADP-enzyme, the NAD-enzyme requires that oxalacetate or pyruvate selectively bind to the protonated form of the enzyme. Both enzymes prefer Mn2+ over Mg2+ for oxalacetate decarboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.     

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.     

Human dihydrofolate reductase: reduction of alternative substrates, pH effects, and inhibition by deazafolates.

The kinetics of the NADPH-dependent reduction of 7,8-dihydrofolate, folate, and 7,8-dihydrobiopterin by human dihydrofolate reductase have been examined over the pH range from 4.0 to 9.5. The V and V/K profiles obtained with the three substrates indicate that a single ionizing residue at the active site of the enzyme must be protonated for catalysis. Both the maximum velocity of the reactions and the rate of interaction of the substrates with the enzyme-NADPH complex decrease in the order dihydrofolate greater than dihydrobiopterin much greater than folate. From the pK values of the V/K profiles, it can be concluded that, while dihydrofolate behaves as a sticky substrate and dihydrobiopterin exhibits slight stickiness, folate is not a sticky substrate. Further support for this conclusion comes from the results of deuterium isotope effects. The pK values obtained from both the V and V/Kfolate profiles are similar to the intrinsic pK value of 5.6 for both the free enzyme and the enzyme-NADPH complex. The folate analogue, 5-deazafolate, is not a substrate, but it undergoes strong interaction with the enzyme. This interaction, which is enhanced by the presence of NADPH, is due to protonation of the bound ligand that does not involve the single ionizing group at the active center of the enzyme. Difference spectra yield evidence for the protonation of bound 5-deazafolate and show that, on binding to the enzyme-NADPH complex, the pK of the N-8 atom is raised to about 10 from a value of about 4 in solution. The results are in accord with those of a recent paper on the three-dimensional structure of the enzyme-5-deazafolate complex [Davies, J.F., Delcamp, T.J., Prendergast, N.J., Ashfors, V.A., Freisheim, J.H., & Kraut, J. (1990) Biochemistry 29, 9467-9479] which indicate that there is hydrogen bond formation between N-8 of the ligand and the carbonyl group of Ile-7. However, the present findings do not support the idea that bound 5-deazafolate resembles the transition-state complex for folate reduction. Quinazolines also interact strongly with the enzyme but in a pH-independent manner. The dissociation constants for the binary complexes are an order of magnitude lower than that for the binding to the enzyme of unprotonated 5-deazafolate. This difference reflects the hydrophobic nature of the amino acid residues at the active site that are near the N-5 and N-8 nitrogens of bound pterins.     

Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine)

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.     

A novel mechanism for substrate inhibition in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase

Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase undergoes significant inhibition of activity with increasing concentrations of its substrate, hydroxypyruvic acid phosphate. The enzyme also displays an unusual dual pH optimum. A significant decrease in the K(i) for substrate inhibition at pH values corresponding to the valley between these optima is responsible for this phenomena. The change in K(i) has an average pK of approximately 5.8 and involves two functional groups that are protonated and two functional groups that are unprotonated for optimal substrate inhibition to occur. Mutagenesis of positively charged amino acid residues at a putative anion binding site previously revealed by the x-ray structure, produces significant changes in the pH-dependent profile of substrate inhibition. Several single residue mutations eliminate the dual pH optima by reducing substrate inhibition between pH 5 and 7 and a triple mutation was identified that eliminates the substrate inhibition altogether. The mutagenesis data support the conclusion that the anion binding site represents a new allosteric site for the control of enzyme activity and functions in a novel mechanism for substrate inhibition.