Steady-state Kinetic Isotope Effects Research Articles

Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants.

Steady-state kinetic isotope effects on enzyme-catalyzed reactions are often interpreted in terms of the microscopic rate constants associated with the elementary reactions of interest. Unfortunately, this approach can lead to confusion, especially when more than one elementary reaction is isotopically sensitive, because it forces one to consider the full catalytic cycle one step at a time rather than as a complete whole. Herein we argue that shifting focus from intrinsic effects to net rate constants and enzyme intermediate concentrations provides a more natural and holistic interpretation by which the effects of partial rate limitation are more easily understood. In doing so, we demonstrate how the experimental determination of isotope effects on enzyme intermediate concentrations allows a direct determination of isotope effects on net rate constants. The chapter is divided into three main sections. The first outlines the basic theory and its interpretation. The second discusses an application of the theory in the study of the radical SAM enzyme DesII. The final section then provides the complete mathematical treatment.

Steady-State Kinetic Isotope Effects Support a Complex Role of Arg226 in the Proposed Desulfonation Mechanism of Alkanesulfonate Monooxygenase

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid-base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O(-) intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.0 ± 0.2 on the kcat parameter, supporting abstraction of the α-proton from the alkane peroxyflavin as the rate-limiting step in catalysis. Previous studies implicated Arg226 as the acid involved in the reprotonation of the hydroxyflavin intermediate. Solvent isotope kinetic studies gave an inverse isotope effect on (D2O)kcat of 0.75 ± 0.04 with no observable effect on (D2O)kcat/Km. This resulted in equivalent solvent isotope effects on (D2O)kcat and (D2O)(kcat)D, suggesting a solvent equilibrium isotope effect on a step occurring after the first irreversible step through product release. Data from proton inventory studies on kcat were best fit to a dome-shaped curve consistent with a conformational change to an open conformation during product release. The solvent isotope effect data coupled with the corresponding proton inventory results support and extend our previous observations that Arg226 donates a proton to the FMN-O(-) intermediate, triggering a conformational change that opens the enzyme to solvation and promotes product release.

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0.22 ± 0.03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1.8 ± 0.2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.

Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase

Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N5-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP+, and not NAD+, protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP+ to also play a role in stabilization of the C4a-hydroperoxyflavin.

Kinetics of the reaction catalyzed by inositol dehydrogenase fromBacillussubtilisand inhibition by fluorinated substrate analogs

Inositol dehydrogenase (EC 1.1.1.18) from Bacillus subtilis catalyzes the oxidation of myo-inositol to scyllo-inosose by transfer of the equatorial hydride of the substrate to NAD+. This is a key enzyme in the metabolism of myo-inositol, a primary carbon source for soil bacteria. In light of our recent discovery that the enzyme has a broad substrate spectrum while maintaining high stereoselectivity, we seek a more thorough understanding of the enzyme and its active site. We have examined the kinetics of the recombinant enzyme, and synthesized fluorinated substrate analogues as competitive inhibitors. We have evaluated all rate constants in the ordered, sequential Bi Bi mechanism. No steady-state kinetic isotope effect is observed using myo-[2-2H]-inositol, indicating that the chemical step of the reaction is not rate-limiting. We have synthesized the substrate analogs 2-deoxy-2-fluoro-myo-inositol, its equatorial analog 1-deoxy-1-fluoro-scyllo-inositol, the gem-difluorinated analog 1-deoxy-1,1-difluoro-scyllo-inositol, and the sugar analog α-D-glucosyl fluoride. Of these, 1-deoxy-1-fluoro-scyllo-inositol showed no inhibition, while all others tested had Kivalues comparable to the Kmvalues of the analogous substrates myo-inositol and α-D-glucose.Key words: inositol dehydrogenase, enzyme mechanism, kinetics, competitive inhibitor, substrate analogue.

Structure and Hydride Transfer Mechanism of a Moderate Thermophilic Dihydrofolate Reductase from Bacillus stearothermophilus and Comparison to Its Mesophilic and Hyperthermophilic Homologues,

Dihydrofolate reductase (DHFR) from a moderate thermophilic organism, Bacillus stearothermophilus, has been cloned and expressed. Physical characterization of the protein (BsDHFR) indicates that it is a monomeric protein with a molecular mass of 18,694.6 Da (0.8), coincident with the mass of 18 694.67 Da calculated from the primary sequence. Determination of the X-ray structure of BsDHFR provides the first structure for a monomeric DHFR from a thermophilic organism, indicating a high degree of conservation of structure in relation to all chromosomal DHFRs. Structurally based sequence alignment of DHFRs indicates the following levels of sequence identity and similarity for BsDHFR: 38 and 58% with Escherichia coli, 35 and 56% with Lactobacillus casei, and 23 and 40% with Thermotoga maritima, respectively. Steady state kinetic isotope effect studies indicate an ordered kinetic mechanism at elevated temperatures, with NADPH binding first to the enzyme. This converts to a more random mechanism at reduced temperatures, reflected in a greatly reduced K(m) for dihydrofolate at 20 degrees C in relation to that at 60 degrees C. A reduction in either temperature or pH reduces the degree to which the hydride transfer step is rate-determining for the second-order reaction of DHF with the enzyme-NADPH binary complex. Transient state kinetics have been used to study the temperature dependence of the isotope effect on hydride transfer at pH 9 between 10 and 50 degrees C. The data support rate-limiting hydride transfer with a moderate enthalpy of activation (E(a) = 5.5 kcal/mol) and a somewhat greater temperature dependence for the kinetic isotope effect than predicted from classical behavior [A(H)/A(D) = 0.57 (0.15)]. Comparison of kinetic parameters for BsDHFR to published data for DHFR from E. coli and T. maritima shows a decreasing trend in efficiency of hydride transfer with increasing thermophilicity of the protein. These results are discussed in the context of the capacity of each enzyme to optimize H-tunneling from donor (NADPH) to acceptor (DHF) substrates.

The interpretation of multiple-step transient-state kinetic isotope effects

In contrast to steady-state kinetic isotope effects (KIEs), transient-state tKIEs are both time and signal dependent and therefore require a very different form of theory for their interpretation. We have previously provided such a theory for the case of single-step isotopic substitutions. No such properly derived theory applicable to the analysis of multiple-step isotopic substitutions required by transient-state solvent isotope effect studies has been available up to this time. Here, we set forth a more general form of that theory which is applicable to multiple-step substituted cases. We prove three theorems: 1. the observed transient-state KIE for any given reactive component in the reaction sequence evaluated at zero time (tKIE 0) is in fact the arithmetic product of the intrinsic KIEs of all the steps that precede the formation of that component. 2. The observed tKIE 0 is completely independent of the intrinsic KIEs of any reverse step in the reaction. 3. The intrinsic KIE of any step may be obtained by dividing the value of the tKIE 0 for that step by the value of the tKIE 0 of the immediately preceding step in the reaction sequence.

Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A.

Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters. To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae. Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction. This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis. The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments. For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are similar in value. Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K(m), and k(red)/K(s) with alpha, alpha-[(2)H]benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis. Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] are in excellent agreement. Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly. The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma). Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations. The positive correlation of rate with sigma (rho approximately 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage. These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B [Walker, M. C., and Edmondson, D. E. (1994) Biochemistry 33, 7088-7098].

Interpretation of transient-state kinetic isotope effects.

In contrast to steady-state kinetic isotope effects (KIE's), transient-state KIE's are dependent on both time and signal source. We developed a theory which predicts the behavior of transient-state KIE's, permits the calculation of the intrinsic KIE, and makes possible the assignment of various optical signals to either pre- or post-hydride transfer events. We proved that the behavior of KIEobs for a reversible two-step reaction for all possible values of the rate constants and all possible ratios of intermediate and product contributions obeys three simple rules (assuming that the isotope-sensitive step involves a hydride transfer): (1) If only the post-hydride species contributes to the observed signal, KIEobs = KIEint at t = 0 and then decreases with time. (2) If only the pre-hydride species contributes to the observed signal, then KIEobs = 1 at t = 0 and then decreases with time. (3) If both pre- and post-hydride species contribute to the observed signal, then KIEobs = 1 at t = 0 and then will either rise or fall with time depending on the relative molar signal coefficients of the pre- and post-hydride species. We provide experimental evidence that the phenomena predicted by this theory do in fact occur in enzyme-catalyzed reactions.

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195–4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indolelyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for α,/β-elimination (except S -ethyl-l-cysteine) exhibited lower kcat (10–30%) and kcat/Km (1–10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for l-phenylalanine and l-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-l-tyrosine with wild-type tyrosine phenol-lyase is identical to that of l-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333–7338]. The pH profile of kcatKm for this reaction exhibits two pKa values with an average of 7.7 ± 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 ± 0.3. However, kcat for 3-fluoro-l-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on fccat and kcat/Km for 3-fluoro-l-[α-2H]-tyrosine are independent of pH and equal to 3.9 ± 0.2 and 2.2±0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 ± 0.2 and 3.8 ± 0.3, respectively. Based on these observations and the pH data, we conclude that α-proton abstraction is apparently the rate-determining step in the reaction of 3-fluoro-l-tyrosine with [H343A]tyrosine phenol-lyase; however, quinonoid intermediate formation (140 s −1) is much faster than kcat of 3-fluoro-L-tyrosine (1.4 s −1) for wild-type tyrosine phenol-lyase; thus, the mutation of His343Ala has apparently changed the rate-determining step of the reaction with 3-fluoro-l-tyrosine. l-Methionine, l-homoserine, l-phenylalanine and l-alanine form quinonoid intermediates with wild-type tyrosine phenol-lyase which exhibit absorbance peaks at about 500 nm but the intensity of these absorbance peaks is dramatically reduced for the mutant. Single-wavelength stopped-flow kinetic measurements demonstrate that [H343A]tyrosine phenol-lyase exhibits slower rate constants for α-deprotonation and faster rate constants for reprotonation than does the wild-type. Taken together, these results demonstrate that, while His343 is not an essential basic group in tyrosine phenol-lyase, it does play an important function in catalysis, possibly by facilitating the conformational change from an ‘open’ to ‘closed’ form when substrates bind.

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R.L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195--4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha, beta-elimination (except S-ethyl-L-cysteine) exhibited lower kcat (10-30%) and kcat/Km (1-10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for L-phenylalanine and L-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-L-tyrosine with wild-type tyrosine phenol-lyase is identical to that of L-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333-7338]. The pH profile of kcat/Km for this reaction exhibits two pKa values with an average of 7.7 +/- 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 +/- 0.3. However, kcat for 3-fluoro-L-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on kcat and kcat/Km for 3-fluoro-L-[alpha-2H]-tyrosine are independent of pH and equal to 3.9 +/- 0.2 and 2.2 +/- 0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 +/- 0.2 and 3.8 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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

Kinetic isotope effects in the oxidation of isotopically labeled NAD(P)H by bacterial flavoprotein monooxygenases.

Three bacterial flavoprotein monooxygenases, p-hydroxybenzoate hydroxylase, orcinol hydroxylase, and salicylate hydroxylase, have been examined for steady-state kinetic isotope effects with (4R)-[4-2H]NAD(P)H and (4R)-[4-3H]NAD(P)H. The observed isotope selections are for deuterium, DV = 1.8-3.5 and D(V/K) = 1.7-5.1, and for tritium, T(V/K) = 5-19. For both orcinol hydroxylase and p-hydroxybenzoate hydroxylase, reduction of enzyme-bound FAD by (4R)-[4-2H]NAD(P)H in pre-steady-state assays reveals intrinsic deuterium isotope effects of 10 +/- 2 on this redox step. These values are at the upper end of substrate deuterium effects seen in enzymatic reactions. Suppression of approximately 83% of the intrinsic isotope effects in the overall reaction rate (e.g., kH/kD = 10 down to DV = 2.5) corroborates earlier kinetic data on p-hydroxybenzoate hydroxylase [Husain, M., & Massey. V. (1979) J. Biol. Chem. 254, 6657] and suggests that these bacterial phenolic monooxygenases balance out internal transition states such that no single barrier is fully rate limiting.