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)     

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.     

Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

Critical ionizing groups in Aeromonas neutral protease

Aeromonas neutral protease possesses two residues critical to its activity. One has a pKa of 5.5 in both the free enzyme and the enzyme-substrate complex and must be deprotonated for maximal activity. The other, which ionizes at pH 7.1 in the free enzyme and at pH 7.4 in the enzyme-substrate complex, must be protonated for optimal enzyme action. The protease is reversibly inhibited by aminoacyl hydroxamates, peptides containing a phenylalanyl residue, phosphoryl-L-phenylalanylglycylglycine, and by beta-phenylpropionyl-L-phenylalanine. The pH dependence of inhibition by the latter revealed that a residue with a pKa of 5.6 influences inhibitor binding. Compounds containing both a hydroxamido group and a chloroacetyl group are particularly effective in inactivating the enzyme, and inhibition is enhanced by hydrophobic residues. Thus, a 33-fold molar excess of chloroacetyl-N-hydroxy-L-phenylalanyl-L-alanyl-L-alanine amide rapidly inactivated Aeromonas neutral protease. Carbethoxylation experiments resulted in a 90% loss in activity which was fully reversible by hydroxylamine; spectral analysis indicated the involvement of a single histidine residue. Protection against both esterification and carbethoxylation was furnished by the presence of beta-phenylproprionyl-L-phenylalanine. Inactivation experiments suggest that a glutamic or aspartic acid and a histidine residue are responsible for the pKa values revealed by pH dependence studies.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

The pH dependence of binding of inhibitors to bovine adrenal tyrosine hydroxylase

The inhibition of purified bovine adrenal tyrosine hydroxylase by several product and substrate analogues has been studied to probe the kinetic mechanism. Norepinephrine, dopamine, and methylcatechol are competitive inhibitors versus tetrahydropterins and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine is an uncompetitive inhibitor versus tetrahydropterins and a competitive inhibitor versus tyrosine. The Ki value for 3-iodotyrosine depends on the tetrahydropterin used. These results are consistent with tetrahydropterin binding first to the free enzyme followed by binding of tyrosine. 5-Deaza-6-methyltetrahydropterin is a noncompetitive inhibitor versus tetrahydropterins and tyrosine. The effect of varying the concentration of tyrosine on the Ki value for 5-deaza-6-methyltetrahydropterin is consistent with the binding of this inhibitor to both the free enzyme and to an enzyme-dihydroxyphenylalanine complex. Dihydroxyphenylalanine also is a noncompetitive inhibitor versus tetrahydropterins and tyrosine; the effect of changing the fixed substrate is consistent with the binding of this inhibitor to both the free enzyme and to the enzyme-tetrahydropterin complex. The effect of pH on the Ki values was determined in order to measure the pKa values of amino acid residues involved in substrate binding. Tight binding of catechols requires that a group with a pKa value of 7.6 be deprotonated. Binding of 3-iodotyrosine involves two groups with pKa values of 7.5 and about 5.5, one of which must be protonated for binding. Binding of 5-deaza-6-methyltetrahydropterin requires that a group on the free enzyme with a pKa value of 6.1 be protonated. The Ki value for dihydroxyphenylalanine is relatively insensitive to pH, but the inhibition pattern changes from noncompetitive to competitive above pH 7.5, consistent with the measured pKa values for binding to the free enzyme and to the enzyme-tetrahydropterin complex.     

The kinetic behavior of chicken liver sulfite oxidase.

A comprehensive kinetic study of sulfite oxidase has been undertaken over the pH range 6.0-10.0, including conventional steady-state work as well as rapid kinetic studies of both the reaction of oxidized enzyme with sulfite and reduced enzyme with cytochrome c (III). A comparison of the pH dependence of kcat, kred, and kox indicates that kred is principally rate limiting above pH 7, but that below this pH the pH dependence of kcat is influenced by that of kox. The pH independence of kred is consistent with our previous proposal concerning the reaction mechanism, in which attack of the substrate lone pair of electrons on a Mo(VI)O2 unit initiates the catalytic sequence. The pH dependence of kred/Kdsulfite indicates that a group on the enzyme having a pKa of approximately 9.3 must be deprotonated for effective reaction of oxidized enzyme with sulfite, possibly Tyr 322, which from the crystal structure of the enzyme constitutes part of the substrate binding site. There is no evidence for the HSO3-/SO32- pKa of approximately 7 in the pH profile for kred/Kdsulfite, suggesting that enzyme is able to oxidize the two equally well. By contrast, kcat/Kmsulfite and kred/Kdsulfite exhibit distinct pH dependence (the former is bell-shaped, the latter sigmoidal), again consistent with the oxidative half-reaction contributing to the kinetic barrier to catalysis at low pH. The pH dependence of kcat/Km(cyt c) (reflecting the second-order rate of reaction of free enzyme with free cytochrome) is bell-shaped and closely resembles that of kox/Kd(cyt c), reflecting the importance of the oxidative half-reaction in the low substrate concentration regime. The pH profile for kox/Kd(cyt c) indicates that two groups with a pKa of approximately 8 are involved in the reaction of free reduced enzyme with cytochrome c, one of which must be deprotonated and the other protonated. These results are consistent with the known electrostatic nature of the interaction of cytochrome c with its physiological partners.     

Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies. Binding of carbamoyl phosphate

Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

The role of amino acid residues in the active site of a midgut microvillar aminopeptidase from the beetle Tenebrio molitor

Aminopeptidases are major enzymes in the midgut microvillar membranes of most insects and are targets of insecticidal Bacillus thuringiensis crystal delta-endotoxins. Sequence analysis and substrate specificity studies showed that these enzymes resemble mammalian aminopeptidase N, although information on the organization of their active site is lacking. The effect of pH at different temperatures on the kinetic parameters of Tenebrio molitor (Coleoptera) larval aminopeptidase showed that enzyme catalysis depend on a deprotonated (pK 7.6; DeltaH degrees (ion), 7.6 kJ/mol) and a protonated (pK 8.2; DeltaH degrees (ion), 16.8 kJ/mol) group. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and diethylpyrocarbonate inactivate the enzyme by modifying a pK 5.8 carboxylate and a imidazole group, respectively, with a reaction order around 1. Tetranitromethane changes the K(m) of the enzyme without affecting its V(max) by modifying a phenol group. The presence of a competitive inhibitor decrease the inactivation reaction rates in all these cases. EDTA inactivation of the aminopeptidase is affected by pH and temperature suggesting the involvement in metal binding of at least one deprotonated imidazole group (pK 5.8, DeltaH degrees (ion), 20 kJ/mol). The data support the hypothesis that T. molitor aminopeptidase catalysis depends on a catalytic metal and on a carboxylate and a protonated imidazole group, whereas substrate binding relies in one phenol and one carboxylate groups. The insect aminopeptidase shares common features with mammalian aminopeptidase N, although differing in details of substrate binding and in residues directly involved in catalysis.     

Role of protonatable groups of bovine heart bc(1) complex in ubiquinol binding and oxidation.

The pH dependence of the initial reaction rate catalyzed by the isolated bovine heart ubiquinol-cytochrome c reductase (bc1 complex) varying decylbenzoquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group with pKa = 5.7 +/- 0.2, while the inhibition constant (Ki) for DB decreased 22 and 2.8 times when groups with pKa = 5.2 +/- 0.6 and 7.7 +/- 0.2, respectively, were protonated. This suggests stabilization of the protonated form of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pKa approximately 5.7 group blocked catalysis, indicating its role in proton transfer. The kinetic model assumed that the deprotonation of two groups (pKa values of 7.5 +/- 0.03 and approximately 9.2) decreases the catalytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) cluster. The protonation of the pKa approximately 7.5 group also decreased the reaction rate by 80-86%, suggesting its role as acceptor of a proton from ubiquinol. The lack of effect on the Km for DBH when the pKa 7.5-7.7 group is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Qo site. The possible relationship of the pKa 5.2-5.7 and pKa 7.5-7.7 groups with Glu272 of cytochrome b and His161 of the Fe-S protein is discussed.     

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.     

Mechanistic aspects of the low-molecular-weight phosphatase activity of the calmodulin-activated phosphatase, calcineurin

Product and substrate analogs have been employed as inhibitors of the low-molecular-weight phosphatase activity of calcineurin, a calmodulin-activated protein phosphatase. Product inhibition kinetics demonstrate that both products, para-nitrophenol and inorganic phosphate, inhibit para-nitrophenyl phosphate hydrolysis in a competitive manner. Inorganic phosphate is a linear competitive inhibitor, whereas the inhibition by para-nitrophenol is more complex. An analog of para-nitrophenol, pentafluorophenol, was found to be a linear competitive inhibitor. These patterns indicate a rapid equilibrium random kinetic mechanism for calcineurin. This mechanism suggests that calcineurin does not generate a phosphoryl enzyme during its catalytic reaction. Application of sulfate analogs indicates that binding of substrate occurs via the phosphoryl moiety. It is suggested that binding is a function of the affinity of ligand for the metal ion involved in calcineurin action. The dependence of the kinetic parameters of calcineurin upon pH was examined to provide information concerning the role of protonation in the activity and specificity of calcineurin. Log (VM) versus pH data for two low-molecular-weight substrates, para-nitrophenyl phosphate and tyrosine-O-phosphate, reveal a pKa value for the enzyme-substrate complex. Analysis of log (VM/KM) data yields a pKa value for the free enzyme of 8.0. Protonation of the phenolic leaving group during hydrolysis is not the rate-limiting step in calcineurin catalysis.     

An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha.

The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding. Br?nsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.     

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)     

pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile. The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile. By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetic mechanism of rat kidney gamma-glutamylcysteine synthetase.

A detailed kinetic investigation was made of the binding mechanism of gamma-glutamylcysteine synthetase purified from rat kidney. The results of initial rate and inhibition studies are consistent with a partially random mechanism in which ATP is the obligatory first substrate and both amino acids bind in a random order to the enzyme-ATP complex. Formation of the enzyme-substrate quaternary complex is necessary prior to release of products. This mechanism is consistent with previous binding studies with the enzyme and while it does not rule out participation of enzyme-bound gamma-glutamyl phosphate as an intermediate in catalysis, such an intermediate cannot be a discrete covalent complex.     

Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.