Determination of the rate-limiting steps and chemical mechanism of fructokinase by isotope exchange, isotope partitioning, and pH studies.

Isotope exchange studies show that beef liver fructokinase has a random kinetic mechanism in which release of fructose from the enzyme is slower than that catalytic reaction. The stickiness of fructose in the presence of MgATP is confirmed by isotope partition studies, which show it to be released 0.53 times as fast as V1/Et in the presence, and 80--130 times as fast in the absence of MgATP. Fructose-1-P release from it binary complex is not at all rate limiting in the forward direction since no exchange of MgADP back into MgATP could be observed during the forward reaction. Failure to find any isotope effect by the equilibrium perturbation method with [1-18O]fructose (upper limit, 1.003, shows that P--O bond cleavage or formation is not rate limiting. The pH profiles for the forward reaction show a group (probably carboxyl with pK 5.7-6.0 and deltaHion = 0) that must be ionized and a group (perhaps lysine, with pK 9--10, and deltaHion 5-9 kcal/mol) which must be protonated for activity. The profile for the back reaction shows only a group with pK 5.5--6 that must be protonated for activity. A chemical mechanism is proposed in which a carboxyl group on the enzyme accepts a proton from the 1-hydroxyl of fructose during the forward reaction and donates it back during the reverse reaction.     

Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects.

Human malic enzyme was studied by steady state kinetics, deuterium isotope effects, and 13C isotope effects with both the physiological dinucleotide cofactor and several alternate cofactors. The log V vs pH profile with NAD revealed two pK(a) values too close to be separately determined, but with an average value of 7.33. The log V/K vs pH profile with NAD revealed two pK(a) values at 7.4 and 5.6. Deuterium and 13C isotope effects indicate that the mechanism of human malic enzyme is stepwise with both NAD and epsilonNAD, but that hyperconjugation in the transition state for hydride transfer is detectable only with the former. With thioNAD and APAD, the isotope effects do not clearly indicate whether the mechanism is stepwise or concerted. The intrinsic 13C isotope effect for decarboxylation was calculated to be 1.0485 by measurement of the partition ratio of oxaloacetate in the presence of NADH and human malic enzyme (decarboxylation to pyruvate/reduction to malate = 2.33). The isotope effect and partitioning data suggest that the energy barrier for decarboxylation of oxaloacetate is not as high relative to the barrier for reduction of oxaloacetate as with the chicken liver enzyme.     

Cytochrome P450 3A4-catalyzed testosterone 6beta-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.     

Transient kinetic studies of malic enzyme. A conformational change associated with substrate inhibition by malate.

The oxidative decarboxylation of l-malate catalyzed by malic enzyme has been studied by stopped-flow spectrophotometry and by initial rate measurements with large concentrations of NADP⁺, malate, and Mn²⁺. The results show that hybride transfer is fast, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 0.7}$ ms. The formation of enzyme-bound NADPH in an amount equivalent to about half of the enzyme active center concentration is followed by turnover at a rate which is initially faster than the steady-state rate, under conditions such that substrate inhibition by malate is observed in the steady state. The steady-state rate is reached after about 0.5 s. It is suggested that a conformational change in the abortive complex of enzyme, manganese, NADPH, and malate is responsible for the malate inhibition and for the slow approach to the true steady state. The relief of malate inhibition by increasing Mn²⁺ concentrations is described, and the results are described in relation to other evidence of nonidentical binding sites for, or negatively cooperative binding of, substrate and activator and possible half-of-the-sites reactivity.     

pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.     

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.     

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

Deuterium isotope effects and 13C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the 13C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the 13C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the 13C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary 13C isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer simulation of primary kinetic isotope effects in the proposed rate-limiting step of the glyoxalase I catalyzed reaction

The proposed rate-limiting step of the glyoxalase I catalyzed reaction is the proton abstraction from the C1 carbon of the substrate by Glu(172). Here we examine primary kinetic isotope effects and the influence of quantum dynamics on this process by computer simulations. The calculations utilize the empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the enzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 +/- 1. 3 is predicted in reasonable agreement with the experimental result of about 3. Furthermore, the magnitude of quantum mechanical effects is found to be very similar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with attaining the required accuracy in order for the present approach to be useful as a diagnostic tool for the study of enzyme reactions are also discussed.     

Triple isotopic labeling and kinetic isotope effects: exposing H-transfer steps in enzymatic systems

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.     

Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.     

Investigations on the kinetic mechanism of octopine dehydrogenase. 2. Location of the rate-limiting step for enzyme turnover.

The kinetic mechanism of octopine dehydrogenase has been investigated by stopped-flow and isotope replacement techniques. When the enzyme is saturated by substrate and coenzyme, both for NADH oxidation and NAD+ reduction, the stationary phase is preceded by a rapid burst. Under these saturation conditions, furthermore, the stationary phase shows a secondary isotope effect when 4S-[4(2)H]NADH is substituted for NADH and when (on the other reaction end) D-[2H] octopine is substituted for D-octopine. The data are taken to indicate that the rate-limiting step for enzyme turnover is a step following a very fast chemical transformation of the reagents. However, when the substrate concentration is lowered below the corresponding Km value keeping the coenzyme concentration at saturating levels, the time course of the reaction shows no burst and the stationary phase has a larger isotope effect. This indicated that under those non-saturating conditions, the enzyme turnover has a larger contribution than the hydrogen-transfer step. Changing the coenzyme concentration alone has very little or no effect on the amplitude of the burst or on the isotope effect. These features are discussed in terms of the other known kinetic properties of the enzyme, and in terms of analogous studies reported in the literature for other dehydrogenases.     

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 use of isotope effects to determine enzyme mechanisms

Isotope effects are one of the most powerful kinetic tools for determining enzyme mechanisms. There are three methods of measurement. First, one can compare reciprocal plots with labeled and unlabeled substrates. The ratio of the slopes is the isotope effect on V/K, and the ratio of the vertical intercepts is the isotope effect on V(max). This is the only way to determine V(max) isotope effects, but is limited to isotope effects of 5% or greater. The second method is internal competition, where the labeled and unlabeled substrates are present at the same time and the change in their ratio in residual substrate or in product is used to calculate an isotope effect, which is that on V/K of the labeled reactant. This is the method used for tritium or (14)C, or with the natural abundances of (13)C, (15)N, or (18)O. The third method involves perturbations from equilibrium when a labeled substrate and corresponding unlabeled product are present at chemical equilibrium. This also gives just an isotope effect on V/K for the labeled reactant. The chemistry is typically not fully rate limiting, so that the isotope effect on V/K is given by: (x)(V/K)=((x)k+c(f)+c(r)(x)K(eq))/(1+c(f)+c(r)) where x defines the isotope (D, T, 13, 15, 18 for deuterium, tritium, (13)C, (15)N, or (18)O), and (x)(V/K), (x)k, and (x)K(eq) are the observed isotope effect, the intrinsic one on the chemical step, and the isotope effect on the equilibrium constant, respectively. The constants c(f) and c(r) are commitments in forward and reverse directions, and are the ratio of the rate constant for the chemical reaction and the net rate constant for release from the enzyme of the varied substrate (direct comparison) or labeled substrate (internal competition and equilibrium perturbation) for c(f), or the first product released or the one involved in the perturbation for c(r). The intrinsic isotope effect, (x)k, can be estimated by comparing deuterium and tritium isotope effects on V/K, or by comparing the deuterium isotope effect with (13)C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the (13)C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a (13)C isotope effect allows one to tell if the two isotope effects are on the same or different steps. Applications of these methods to several enzyme systems will be presented.