Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.     

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.     

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.     

Secondary 18O isotope effects for hexokinase-catalyzed phosphoryl transfer from ATP

Secondary 18O isotope effects in the gamma-position of ATP have been measured on phosphoryl transfer catalyzed by yeast hexokinase in an effort to deduce the structure of the transition state. The isotope effects were measured by the remote-label method with the exocyclic amino group of adenine as the remote label. With glucose as substrate, the secondary 18O isotope effect per 18O was 0.9987 at pH 8.2 and 0.9965 at pH 5.3, which is below the pK of 6.15 seen in the V/K profile for MgATP. With the slow substrate 1,5-anhydro-D-glucitol, the value was 0.9976 at pH 8.2. While part of the inverse nature of the isotope effect may result from an isotope effect on binding, the more inverse values when catalysis is made more rate limiting by decreasing the pH or switching to a slower substrate suggest a dissociative transition state for phosphoryl transfer, in agreement with predictions from model chemistry. The 18O equilibrium isotope effect for deprotonation of HATP3- is 1.0156, while Mg2+ coordination to ATP4- does not appear to be accompanied by an 18O isotope effect larger than 1.001.     

Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase.

African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 2. Physiological substrates and phosphoenol-alpha-ketobutyrate

pH profiles have been determined for the reactions catalyzed by pyruvate kinase between pyruvate and MgATP and between phosphoenolpyruvate and MgADP. V, V/KMgATP, and V/Kpyruvate all decrease below a pK of 8.3 and above one of 9.2. The group with pK = 8.3 is probably a lysine that removes the proton from pyruvate during enolization, while the pK of 9.2 is that of water coordinated to enzyme-bound Mg2+. The fact that this pK shows in all three pH profiles shows that pyruvate forms a predominantly second sphere complex and cannot replace hydroxide to form the inner sphere complex that results in enolization and subsequent phosphorylation. On the basis of the displacement of the pK of the acid-base catalytic group in its V/K profile, phosphoenolpyruvate is a sticky substrate, reacting to give pyruvate approximately 5 times faster than it dissociates. The V/K profile for the slow substrate phosphoenol-alpha-ketobutyrate shows the pK of 8.3 for the acid-base catalytic group in its correct position, but this group must be protonated so that it can donate a proton to the intermediate enolate following phosphoryl transfer. The secondary phosphate pK of the substrate is seen in this V/K profile as well as in the pKi profile for phosphoglycolate (but not in those for glycolate O-sulfate or oxalate), showing a preference for the trianion for binding. The chemical mechanism with the natural substrates thus appears to involve phosphoryl transfer between MgADP and a Mg2+-bound enolate with metal coordination of the enolate serving to make it a good leaving group.     

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.     

Investigation of substrate specificity of creatine kinase using chromium (III) and cobalt(III) complexes of adenosine 5'-diphosphate

The specificity for substrate binding to creatine kinase for metal-nucleotide complexes of the type Cr-(H2O)4-n(NH3)nADP (where n = 0, 3, or 4) and Co-(H2O)4-m(NH3)mADP (for m = 3 or 4) has been investigated over the pH range 5.5-7.8 with the delta-alpha, beta-bidentate diastereoisomers. These inert nucleotide complexes acted as competitive inhibitors vs. MgADP over this range. In addition, the pH dependence of the V, V/K, and Km values for MgADP has been determined. Metal-nucleotide binding to the enzyme is strongest below an approximate pK of 6.45 but again becomes pH independent above pH 7. This pK is not associated with the metal-nucleotide complex. Instead, we conclude that the pK of the acid-base catalyst (thought to be histidine) is about 6.45 in the absence of nucleotide but is raised to 7.2 in its presence. This perturbation of the pK may result from a protein conformational change that allows a hydrogen bond to form between the phosphorylated nitrogen of phosphocreatine and the acid-base catalyst. The pK of the water in Cr(H2O)(NH3)3ADP has been determined to be 6.6, and by comparison of the binding affinity of this complex with that of Cr(NH3)4ADP or Cr(H2O)4ADP, it can be deduced that the hydroxo species binds more strongly than the aquo complex. In general, chromium nucleotides are bound more strongly than cobalt complexes, and binding affinity increases as water replaces ammonia in the first coordination sphere of the metal. Both trends are a result of stronger hydrogen-bond interactions between the metal complex and protein.     

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.     

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 1. Alternate substrates oxalacetate, glycolate, hydroxylamine, and fluoride

The decarboxylation of oxalacetate shows equilibrium-ordered kinetics, with Mg2+ adding before oxalacetate. The Ki for Mg2+ increases below a pK of 6.9, corresponding to a ligand of the metal that is probably glutamate, and decreases above a pK of 9.2, corresponding to water coordinated to enzyme-bound Mg2+. Both V and V/KOAA decrease above the pK of 9.2, suggesting that the carbonyl oxygen of oxalacetate must replace water in the inner coordination sphere of Mg2+ prior to decarboxylation. The enzyme-Mg2+-oxalacetate complex must be largely an outer sphere one, however, since the pK of 9.2 is seen in the V profile. The phosphorylation of glycolate or N-hydroxycarbamate (the actual substrate that results from reaction of hydroxylamine with bicarbonate) occurs only above the pK of 9.2, with V/K profiles decreasing below this pH. The alkoxides of these substrates appear to be the active species, replacing water in the coordination sphere of Mg2+ prior to phosphorylation by MgATP. Glycolate, but not N-hydroxycarbamate, can bind when not an alkoxide, since the V profile for the former decreases below a pK of 8.9, while V for the latter is pH independent. Initial velocity patterns for phosphorylation of fluoride in the presence of bicarbonate show saturation by MgATP but not by fluoride. The V/K profile for fluoride decreases above the pK of 9.0, showing that fluoride must replace water in the coordination sphere of Mg2+ prior to phosphorylation. None of the above reactions is sensitive to the protonation state of the acid-base catalyst that assists the enolization of pyruvate in the physiological reaction.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

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.     

Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

We have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally less than 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. We conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of approximately 9 from the pKi profile. Quantitative analysis of the isotope effects suggests an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate of approximately 1.03 for both substrates; however, the partition ratio of this intermediate for release of ammonia as opposed to back-reaction is 14 times greater for adenosine (1.4) than for 7,8-dihydro-8-oxoadenosine (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase

Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co2+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the V for oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the pKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mg2+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity.     

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.     

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7-9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV-visible (UV-vis) absorption and resonance Raman (RR). In 7-9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis-His-ligated heme center. In acidic 9 M urea solutions the UV-vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H(2)O complex. The pK(a) for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the pK(a) 4.8. The formal redox potential of the His/H(2)O complex of cytochrome c in 9 M urea at pH 3 was estimated to be -0.13 V, ca. 100 mV more positive than E degrees ' estimated for the bis-His complex of cytochrome c in urea solution at pH 7.     

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)     

Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase

The catalytic mechanism of porcine heart NADP isocitrate dehydrogenase has been investigated by use of the variation of deuterium and 13C kinetic isotope effects with pH. The observed 13C isotope effect on V/K for isocitrate increases from 1.0028 at neutral pH to a limiting value of 1.040 at low pH. The limiting 13C isotope effect with deuteriated isocitrate at low pH is 1.016. This decrease in 13(V/KIc) upon deuteriation indicates a stepwise mechanism for the oxidation and decarboxylation of isocitrate. This predicts a deuterium isotope effect on V/K of 2.9, but D(V/K) at low pH only increases to a maximum of 1.08. It is not known why 13(V/KIc) with deuteriated isocitrate decreases more than predicted. The pK seen in the 13(V/KIc) pH profile for isocitrate is 4.5. This pK is displaced 1.2 pH units from the true pK of the acid/base functionality of 5.7 seen in the pKi profile for oxalylglycine, a competitive inhibitor for isocitrate. From this displacement, catalysis is estimated to be 16 times faster than substrate dissociation. By use of the pH-dependent partitioning ratio of the reaction intermediate oxalosuccinate between decarboxylation to 2-ketoglutarate and reduction to isocitrate, the forward commitment to catalysis for decarboxylation was determined to be 7.3 at pH 5.4 and 3.2 at pH 5.0. This gives an intrinsic 13C isotope effect for decarboxylation of 1.050. 3-Fluoroisocitrate is a new substrate oxidatively decarboxylated by NADP isocitrate dehydrogenase. At neutral pH, D(V/K3-F-Ic) = 1.45 and 13(V/K3-F-Ic) = 1.0129. At pH 5.2, 13(V/K3-F-Ic) increases to 1.0186, indicating that a finite, but diminished, external commitment remains at neutral pH. The product of oxidative decarboxylation of 3-hydroxyisocitrate by NADP isocitrate dehydrogenase is 2-hydroxy-3-ketoglutarate. This results from enzymatic protonation of the cis-enediol intermediate at C2 rather than C3 (as seen with isocitrate and 3-fluoroisocitrate). 2-Hydroxy-3-ketoglutarate further decarboxylates in solution to 2-hydroxy-3-ketobutyrate, which further decarboxylates to acetol. This makes 3-hydroxyisocitrate unsuitable for 13C isotope effect studies.     

Kinetic studies with myo-inositol monophosphatase from bovine brain

The kinetic properties of myo-inositol monophosphatase with different substrates were examined with respect to inhibition by fluoride, activation or inhibition by metal ions, pH profiles, and solvent isotope effects. F- is a competitive inhibitor versus 2'-AMP and glycerol 2-phosphate, but noncompetitive (Kis = Kii) versus DL-inositol 1-phosphate, all with Ki values of approximately 45 microM. Activation by Mg2+ follows sigmoid kinetics with Hill constants around 1.9, and random binding of substrate and metal ion. At high concentrations, Mg2+ acts as an uncompetitive inhibitor (Ki = 4.0 mM with DL-inositol 1-phosphate at pH 8.0 and 37 degrees C). Activation and inhibition constants, and consequently the optimal concentration of Mg2+, vary considerably with substrate structure and pH. Uncompetitive inhibition by Li+ and Mg2+ is mutually exclusive, suggesting a common binding site. Lithium binding decreases at low pH with a pK value of 6.4, and at high pH with a pK of 8.9, whereas magnesium inhibition depends on deprotonation with a pK of 8.3. The pH dependence of V suggests that two groups with pK values around 6.5 have to be deprotonated for catalysis. Solvent isotope effects on V and V/Km are greater than 2 and 1, respectively, regardless of the substrate, and proton inventories are linear. These results are consistent with a model where low concentrations of Mg2+ activate the enzyme by stabilizing the pentacoordinate phosphate intermediate. Li+ as well as Mg2+ at inhibiting concentrations bind to an additional site in the enzyme-substrate complex. Hydrolysis of the phosphate ester is rate limiting and facilitated by acid-base catalysis.