Oxidation-reduction of general acyl-CoA dehydrogenase by the butyryl-CoA/crotonyl-CoA couple. A new investigation of the rapid reaction kinetics

Pig kidney general acyl-CoA dehydrogenase (GAD) can be reduced by butyryl-CoA to form reduced enzyme and crotonyl-CoA. This reaction is reversible. Stopped-flow, kinetic investigations on GAD have been made, using the following reaction pairs: oxidized GAD/butyryl-CoA, oxidized GAD/crotonyl-CoA, oxidized GAD/alpha,beta-dideuteriobutyryl-CoA, reduced GAD/butyryl-CoA, and reduced GAD/crotonyl-CoA (in 50 mM potassium phosphate buffer, pH 7.6 at 4 degrees C). Reduction of GAD by butyryl-CoA is triphasic. The slowest phase is 100-fold slower than the preceding phase and appears to represent a secondary process not directly related to the primary reduction events. The first two fast phases are responsible for reduction of GAD. Reduction proceeds via a reduced enzyme/crotonyl-CoA charge-transfer complex. alpha, beta-Dideuteriobutyryl-CoA elicits a major deuterium isotope effect (15-fold) on the reduction reaction. Oxidation of GAD by crotonyl-CoA is biphasic. Oxidation proceeds via the same reduced enzyme/crotonyl-CoA charge-transfer complex seen during reduction. The oxidation reaction ends in a mixture composed largely of oxidized GAD species. From the data, we constructed a mechanism for the reduction/oxidation of GAD by butyryl-CoA/crotonyl-CoA. This mechanism was then used to simulate all of the observed kinetic time course data, using a single set of kinetic parameters. A close correspondence between the observed and simulated data was obtained.     

Kinetic methods for the study of the enzyme systems of β-oxidation

Kinetic methods for studying the reactions of the “general” fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive half-reaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the K_m for substrate and K_I for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate.     

Oxidation-reduction potentials of butyryl-CoA dehydrogenase

In order to obtain butyryl-CoA dehydrogenase from Megasphaera elsdenii in pure enough form to perform redox studies, the existing purification procedures first had to be modified and clarified [Engel, P. (1981) Methods Enzymol. 71, 359-366]. These modifications are described, and the previously unpublished spectral properties of the electrophoretically pure CoA-free butyryl-CoA dehydrogenase are presented. In our spectral reductive titration of pure enzyme, we show that although blue neutral flavin radical is stabilized in nonquantitative amounts in dithionite titrations (19%) or in electrochemical reductions mediated by methylviologen (5%), it is not thermodynamically stabilized; therefore, only a midpoint potential for butyryl-CoA dehydrogenase is obtained. The electron-transfer behavior from pH 5.5 to pH 7.0 indicates reversible two-electron transfer accompanied by one proton: EFlox + 2e- + H+ = EFlredH- Em7 = -0.079 V vs. SHE where EFlox is oxidized butyryl-CoA dehydrogenase, EFlredH- is two electron reduced enzyme, and Em7 is the midpoint potential at pH 7.0 at 25 degrees C. Redox data and activity data both indicate that the enzyme loses activity rapidly at pH values above 7.0. The Em7 of the butyryl-CoA dehydrogenase is 40 mV positive of the Em7 of the butyryl-CoA/crotonyl-CoA couple [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. Binding of substrate analogue acetoacetyl-CoA caused the potential of butyryl-CoA dehydrogenase to shift 100 mV negative of the free enzyme. The negative shift in potential makes electron transfer from enzyme to substrate more probable, which is consistent with the direction of electron transfer in the bacterial system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism

A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-[4-2H]NADH and (4R)-[4-3H]NADH as substrates. The DV effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD+ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small (approximately 2-fold) preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex.     

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.     

Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450

Phenylacetylene and biphenylacetylene are oxidized by cytochrome P-450 to the corresponding arylacetic acids. The acetylenic hydrogen shifts to the adjacent carbon and one atom of molecular oxygen is incorporated into the carboxylic acid group in these transformations, which are subject to a large kinetic isotope effect when the acetylenic hydrogen is replaced by deuterium. The same products and isotope effects are observed when the two arylacetylenes are oxidized by m-chloroperbenzoic acid rather than by the enzyme. In contrast, the inactivation of cytochrome P-450 that occurs during the oxidation of phenylacetylene is insensitive to deuterium substitution. The partition ratio between metabolite formation and enzyme inactivation consequently changes from 26 to 15 in going from phenylacetylene to the deuterated analogue. Metabolite formation therefore diverges from heme alkylation very early in the catalytic process.     

The onset of the deuterium isotope effect in cytochrome c oxidase

We have investigated the dynamics of proton equilibration within the proton-transfer pathway of cytochrome c oxidase from bovine heart that is used for the transfer of both substrate and pumped protons during reaction of the reduced enzyme with oxygen (D-pathway). The kinetics of the slowest phase in the oxidation of the enzyme (the oxo-ferryl --> oxidized transition, F --> O), which is associated with proton uptake, were studied by monitoring absorbance changes at 445 nm. The rate constant of this transition, which is 800 s(-)(1) in H(2)O (at pH approximately 7.5), displayed a kinetic deuterium isotope effect of approximately 4 (i.e., the rate was approximately 200 s(-)(1) in 100% D(2)O). To investigate the kinetics of the onset of the deuterium isotope effect, fully reduced, solubilized CO-bound cytochrome c oxidase in H(2)O was mixed rapidly at a ratio of 1:5 with a D(2)O buffer saturated with oxygen. After a well-defined time period, CO was flashed off using a short laser flash. The time between mixing and flashing off CO was varied within the range 0. 04-10 s. The results show that for the bovine enzyme, the onset of the deuterium isotope effect takes place within two time windows of O transition is internal proton transfer from a site, proposed to be Glu (I-286) (R. sphaeroides amino acid residue numbering), to the binuclear center. The spontaneous equilibration of protons/deuterons with this site in the interior of the protein is slow (approximately 1 s).     

Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral and electrochemical properties of glutaryl-CoA dehydrogenase from Paracoccus denitrificans

Studies of the spectral (UV/vis and resonance Raman) and electrochemical properties of the FAD-containing enzyme glutaryl-CoA dehydrogenase (GCD) from Paracoccus denitrificans reveal that the properties of the oxidized enzyme (GCDox) appear to be invariant from those properties known for other acyl-CoA dehydrogenases such as mammalian general acyl-CoA dehydrogenase (GACD) and butyryl-CoA dehydrogenase (BCD) from Megasphaera elsdenii. However, when either free or complexed GCD is reduced, its spectral and electrochemical behavior differs from that of both GACD and BCD. Free GCD does not stabilize any form of one-electron-reduced GCD, but when GCD is complexed to its inhibitor, aceto-acetyl-CoA, the enzyme stabilizes 20% of the blue neutral radical form of FAD (FADH.) upon reduction. Like GACD, when crotonyl-CoA- (CCoA) bound GCD is reduced, the red anionic form of FAD radical (FAD.-) is stabilized, and excess reduction equivalents are necessary to effect full reduction of the complex. A comproportionation reaction is proposed between fully reduced crotonyl-CoA-bound GCD (GCD2e-CCoA) and GCDox-CCoA to partially explain the stabilization of GCD-bound FAD.- by CCoA. When GCD is reduced by its optimal substrate, glutaryl-CoA, a two-electron reduction is observed with concomitant formation of a long-wavelength charge-transfer band. It is proposed that the ETF specific for GCD abstracts one electron from this charge-transfer species and this is followed by the decarboxylation of the oxidized substrate. At pH 6.4, potential values measured for free GCD and GCD bound to acetoacetyl-CoA are -0.085 and -0.129 V, respectively. Experimental evidence is given for a positive shift in the reduction potential of GCD when the enzyme is bound to a 1:1 mixture of butyryl-CoA and CCoA. However, significant GCD hydratase activity is observed, preventing quantitation of the potential shift.     

Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.

Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.     

Pig heart lipoamide dehydrogenase: solvent equilibrium and kinetic isotope effects.

Lipoamide dehydrogenase is a flavoprotein which catalyzes the reversible oxidation of dihydrolipoamide, Lip(SH)2, by NAD+. The ping-pong kinetic mechanism involves stable oxidized and two-electron-reduced forms. We have investigated the rate-limiting nature of proton transfer steps in both the forward and reverse reactions catalyzed by the pig heart enzyme by using a combination of alternate substrates and solvent kinetic isotope effect studies. With NAD+ as the variable substrate, and at a fixed, saturating concentration of either Lip(SH)2 or DTT, inverse solvent kinetic isotope effects of 0.68 +/- 0.05 and 0.71 +/- 0.05, respectively, were observed on V/K. Solvent kinetic isotope effects on V of 0.91 +/- 0.07 and 0.69 +/- 0.02 were determined when Lip(SH)2 or DTT, respectively, was used as reductant. When Lip(SH)2 or DTT was used as the variable substrate, at a fixed concentration of NAD+, solvent kinetic isotope effects of 0.74 +/- 0.06 and 0.51 +/- 0.04, respectively, were observed on V/K for these substrates. Plots of the kinetic parameters versus mole fraction D2O (proton inventories) were linear in all cases. Solvent kinetic isotope effect measurements performed in the reverse direction using NADH as the variable substrate showed equivalent, normal solvent kinetic isotope effects on V/KNADH when oxidized lipoamide, lipoic acid, or DTT were present at fixed, saturating concentrations. Solvent kinetic isotope effects on V were equal to 1.5-2.1. When solvent kinetic isotope effect measurements were performed using the disulfide substrates lipoamide, lipoic acid, or DTT as the variable substrates, normal kinetic isotope effects on V/K of 1.3-1.7 were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the butyryl-CoA dehydrogenase by substrate and product binding

Until now, workers in the field of fatty acid metabolism have suggested that the substrates are isopotential with the enzymes and that the reactions are forced to completion by the formation of charge-transfer complexes [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. To date, no experimental evidence for this hypothesis exists. The work presented here shows that the butyryl-CoA/crotonyl-CoA couple is not isopotential with the enzymes with which it interacts. The potential of the butyryl-CoA/crotonyl-CoA couple (E ' = -0.013 V) is significantly more positive than the potential of either of the enzymes with which it interacts, bacterial butyryl-CoA dehydrogenase (E ' = -0.079 V) and mammalian general acyl-CoA dehydrogenase (E ' = 0.133 V). These data imply that the regulation of enzyme potential is essential for any electron transfer from substrate to enzyme to occur in mammalian or bacterial systems. In support of this assertion, a significant shift in potential for bacterial butyryl-CoA dehydrogenase (an analogue of the mammalian enzyme) in the presence of butyryl-CoA and crotonyl-CoA is reported. The potential is shifted positive by 60 mV. Larger potential shifts will undoubtedly be observed with the mammalian enzyme, which would be consistent with the catalytic direction of electron transfer.     

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.     

Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.     

Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids

A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation. On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined. The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S. cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively. A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes. The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence. Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033). The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction. In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.     

Reactions at prochiral centers. Interdependence in the estimation of enzyme stereospecificity toward prochiral centers and the configurational purity of labeled substrates.

In many enzymic reactions one or the other hydrogen is released from a prochiral center abCH2. To determine the stereospecificity with respect to such centers under nonreversal conditions a substrate with a chiral center abC1HH is employed (H = 3H or 2H). Assuming the simplest possible model, the R specificity of an enzyme or enzyme system (eR) may be defined in terms of the configurational R purity of the substrate with respect to the labeled center (pR), and the fraction of H released (r) or conserved (l - r) during the reaction by the equation eR = [(l - r) -pR]/[1 - 2pR]. Where r is constant, this is a rectangular hyperbola. The apparent stereospecificity calculated with this equation serves as a characteristic of the experimental system irrespective of the assumed model. The problems arising from direct isotope effects are discussed and the theoretical advantages of using product 3H:14C ratios under low conversion conditions noted. Applications of the equation to the determination of configurational purity at labeled centers, to the study of stereospecificity in enzymes and enzyme systems, and to the study of the biosynthesis of natural products are discussed.     

The kinetic mechanism of D-amino acid oxidase with D-alpha-aminobutyrate as substrate. Effect of enzyme concentration on the kinetics

The kinetic mechanism of hog kidney D-amino acid oxidase with D-alpha-aminobutyrate as substrate has been examined in detail using a combination of steady state and rapid reaction methods. At concentrations of D-alpha-aminobutyrate below 0.5 mM, the rapid reaction and steady state results are consistent with the mechanism previously proposed for D-alanine (Massey, V., and Gibson, Q. H. (1964) Fed. Proc. 23, 18-29; Porter, D. J. T., Voet, J. G., and Bright, H. J. (1977) J. Biol. Chem. 252, 4464-4473). Both flavin reduction by D-alpha-aminobutyrate and reoxidation are quite rapid. Release of product from the oxidized enzyme has been measured directly and matches the turnover number at infinite concentrations of both substrates. Substitution of deuterium for the alpha-hydrogen decreases the rate of reduction 1.4-fold, without any effect on the apparent Kd. Computer simulations show that the kinetic isotope effects on the reductive half-reaction with D-alanine reported by Porter et al. (see above reference) can be explained using a two-step model with a kinetic isotope effect of 1.75 on the limiting rate of reduction. The effect of enzyme concentration on the kinetics has been examined in some detail. With D-alanine as substrate, increasing the enzyme concentration over the range 29 nM to 17 microM resulted in less than a 2-fold decrease in the turnover number. The Kd for benzoate binding also decreased marginally with increasing enzyme concentration. The effect of enzyme concentration is consistent with a decrease in the rate of release of ligands from the oxidized enzyme as the enzyme concentration is increased.     

Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase.

The effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme A dehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160 and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemical properties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engage in favorable pi-sigma interactions with the isoalloxazine ring of the flavin to help stabilize formation of the anionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine (F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (E(ox/hq)) to shift negative by 44-54 mV. The E(ox/hq) value was also found to decrease when aromatic residues containing electron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mV for the F160Y and F160W mutants, respectively, are attributed to increased pi-pi repulsive interactions between the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potential for the butyryl-CoA/crotonyl-CoA couple (E(BCoA/CCoA)) to shift 14 mV negative, stabilizing the oxidized product. Formation of product was found to be even more favorable in complexes with the F160Y and F160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.