Identification and role of ionizing functional groups at the active center of Rhodotorula gracilis D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is a flavoprotein oxidase that catalyzes the oxidation of amino acids and produces ketoacids and H(2)O(2). The rate of product release from reduced DAAO from Rhodotorula gracilis is pH dependent and reflects a pK(a) of approximately 9.3. Binding of benzoate and 3,3,3-trifluoro-D-alanine to wild-type and Y238F-DAAO is also pH dependent (pK(a)=9.8+/-0.1 and 9.05+/-0.1, respectively for benzoate binding). However, binding of benzoate to Y223F-DAAO is pH independent, indicating the pK(a) is due to Y223-OH. This latter residue is thus involved in substrate binding, and probably is the group that governs product release. In contrast to this, the second active site tyrosine, Y238, has little influence on ligand binding.

pH and kinetic isotope effects in d-amino acid oxidase catalysis.

The effects of pH, solvent isotope, and primary isotope replacement on substrate dehydrogenation by Rhodotorula gracilis d-amino acid oxidase were investigated. The rate constant for enzyme-FAD reduction by d-alanine increases approximately fourfold with pH, reflecting apparent pKa values of approximately 6 and approximately 8, and reaches plateaus at high and low pH. Such profiles are observed in all presteady-state and steady-state kinetic experiments, using both d-alanine and d-asparagine as substrates, and are inconsistent with the operation of a base essential to catalysis. A solvent deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with d-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary substrate isotope effect on the reduction rate with [2-D]d-alanine is 9.1 +/- 1.5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with [2-D]d-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) are independent of the presence of the other isotope, i.e. the 'double' kinetic isotope effect is the product of the individual KIEs, consistent with a transition state in which rupture of the two bonds of the substrate to hydrogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in d-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pKa,app of approximately 8 is interpreted to arise from the microscopic ionization of the substrate amino acid alpha-amino group, but also includes contributions from kinetic parameters.

Two similar gene clusters coding for enzymes of a new type of aerobic 2-aminobenzoate (anthranilate) metabolism in the bacterium Azoarcus evansii.

In the beta-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a beta-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by beta-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.

Structure and characterization of the glycan moiety of L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

Ophidian L-amino-acid oxidase (L-amino-acid oxygen:oxidoreductase, deaminating, EC 1.4.3.2) is found in the venom of many poisonous snakes (crotalids, elapids and viperids). This FAD-dependent glycoprotein has been studied from several snake species (e.g. Crotalus adamanteus, Crotalus atrox and Calloselasma rhodostoma) in detail with regard to the biochemical and enzymatic properties. The nature of glycosylation, however, as well as the chemical structure(s) of the attached oligosaccharide(s) are unknown. In view of the putative involvement of the glycan moiety in the biological effects of ophidian L-amino-acid oxidase, notably the apoptotic activity of the enzyme, structural knowledge is needed to evaluate its exact function. In this study we report on the glycosylation of L-amino-acid oxidase from the venom of the Malayan pit viper (Calloselasma rhodostoma). Its glycosylation is remarkably homogeneous with the major oligosaccharide accounting for approximately 90% of the total sugar content. Based on detailed analysis of the isolated oligosaccharide by 2D NMR spectroscopies and MALDI-TOF mass spectrometry the glycan is identified as a bis-sialylated, biantennary, core-fucosylated dodecasaccharide. The biological significance of this finding is discussed in light of the biological activities of the enzyme.

Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair.

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-A resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu(475), located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.

Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties.

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.

L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme.

Here we report the cDNA-deduced amino-acid sequence of L-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, D-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl(-), prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.

The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.

Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum.

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.

The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site.

The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO-AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals significant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.

Cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum: effect of surfactants and organic solvents on activity.

We have studied systematically the effect of the non-ionic surfactants Thesit and Triton X-100, and of propan-2-ol (used as a substrate solubilizer) on the activity of the cholesterol oxidases from Streptomyces hygroscopicus (SCO) and Brevibacterium sterolicum (BCO). Low concentrations of Thesit lead to an activity increase with both enzymes; at higher surfactant concentrations the opposite effect occurs. Triton X-100 inactivates both enzymes at all concentrations. It is deduced that these surfactants exert their effects by interaction with the enzymes and not by affecting micellar phenomena. The effect of propan-2-ol on SCO, in contrast with that on BCO, depends on the buffer concentration (potassium phosphate). Other organic solvents induce results similar to those obtained with SCO and propan-2-ol. A significant difference between the two cholesterol oxidases emerges when stability is tested at 25 degrees C and in the presence of different concentrations of propan-2-ol: BCO activity is rapidly inactivated, whereas SCO still has 70% of the initial activity after 5 h in the presence of 30% propan-2-ol. From our results, SCO seems to be the catalyst of choice in comparison with BCO for the exploitation of cholesterol oxidases in biotechnology and applied biochemistry.

Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.

The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s-1 and 105 s-1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s-1 for BCO and 232 s-1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Delta5-6-->Delta4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s-1). With oxidized SCO and BCO the rate of isomerization is much faster ( approximately 300 s-1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates approximately 250 s-1 and 1.3 s-1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme-product complexes.

NIH shift in flavin-dependent monooxygenation: mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase.

The flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase from the eubacterium Azoarcus evansii catalyzes the dearomatization of 2-aminobenzoyl-CoA. The reaction consists in an O2-dependent monooxygenation at the benzene position 5, which is followed immediately by an NADH-dependent hydrogenation of the intermediate at the same catalytic locus. The reaction was studied by 1H, 2H, and 13C NMR spectroscopy of the products. The main product was characterized as 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA by two-dimensional NMR spectroscopy. Thus, [5-2H]2-aminobenzoyl-CoA was converted into [6-2H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 --> 6 shift of the [5-2H] label. Label from NAD2H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D2O was incorporated into the 4 and the 6 positions of 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA. The labeling pattern is compatible with the monooxygenation proceeding via what is formally an NIH shift, yielding 5-oxo-2-aminocyclohex-1, 3-diene-1-carboxyl-CoA as a protein-bound intermediate. It is suggested that this shift in flavin-dependent monooxygenation may have general validity.

Studies with lysine N6-hydroxylase. Effect of a mutation in the assumed FAD binding site on coenzyme affinities and on lysine hydroxylating activity.

The proposed FAD binding site of L-lysine N6-hydroxylase (EC 1.14.13.99) exhibits an unusual proline in a position where a highly conserved glycine is found in other FAD dependent hydroxylases. We have studied the role of this proline by mutating it to glycine in [P14G]aerA, which was expressed in Escherichia coli M15-2 and purified to homogeneity. The mutation has marked effects on the affinities of the cofactors FAD and NADPH as well as the substrate, lysine. Compared to the wild-type enzyme, the activity vs. pH profile of the mutant protein indicates a shift of the apparent pK'(a)s (7.8 and 8.7 for wild-type and 6.8 and 7.7 for the P14G-mutant enzyme) and of the activity maximum (pH 8 for wild-type and pH 7 for the P14G-mutant enzyme). While the activity of the mutant enzyme is much lower under conditions found to be optimal for the wild-type enzyme, adjustment of substrate and cofactor concentrations and pH leads to comparable activities for the mutant enzyme. These results suggest that the proline fulfils an important structural role in the proposed FAD binding site.

Mechanism of activation of acyl-CoA substrates by medium chain acyl-CoA dehydrogenase: interaction of the thioester carbonyl with the flavin adenine dinucleotide ribityl side chain.

The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acyl-coenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3'-deoxy-FAD and ribityl-2'-deoxy-FAD. 3'-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3'-OH group does not play any particular role in cofactor binding or catalysis. 2'-Deoxy-FAD-MCADH was characterized using the natural substrate C8CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2'-deoxy-FAD-MCADH is approximately 1.5 x 10(7)-fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2'-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to approximately 38 kJ mol-1. The alphaC-H microscopic pKa of the substrate analogue 3S-C8CoA, which undergoes alpha-deprotonation on binding to MCADH, is lowered from approximately 16 in the free state to approximately 11 (+/-0.5) when bound to 2'-deoxy-FAD-MCADH. This compares with a decrease of the same pKa to approximately 5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of approximately 11 pK units, i.e., approximately 65 kJ mol-1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of approximately 6 pK units ( approximately 35 kJ mol-1) between MCADH and 2'-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2'-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of alphaC-H microscopic pKa of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2'-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2'-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2'-OH hydrogen bond. The large energy associated with the 2'-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.

Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site.

Medium-chain acyl-CoA dehydrogenase (MCADH) deficiency, an autosomal recessive inherited disorder, is the most common genetic disorder in mitochondrial beta-oxidation in humans. In addition to one prevalent disease-causing mutation (K304E), a series of rarer mutations has been reported, but none of these has yet been characterized in detail. We report here on the biochemical characterization of the purified recombinant mutant protein in which threonine is replaced by alanine at position 168 of the mature protein (T168A-MCADH). It is the first mutation to be found in patients that is located in the active site of the enzyme. Thr-168 is hydrogen-bonded to the flavin N(5) of the cofactor FAD. The thermostability of T168A-MCADH is markedly decreased compared with human wild-type MCADH (hwt-MCADH). Catalytic activity with ferricenium as acceptor is lowered by 80% and with the natural acceptor electron-transferring flavoprotein by over 90% compared with hwt-MCADH. In the mutant the extent of flavin semiquinone formed on reduction is approx. 50% that of hwt-MCADH. The pK reflected by the pH-dependence of Vmax is shifted from approx. 8.2 (hwt-MCADH) to approx. 7 (T168A-MCADH) and the rates of enzyme flavin reduction (stopped-flow measurements) are only approx. 1/10 those of the parent enzyme. These properties are discussed in the light of the possible mechanisms leading to disease in humans.

Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase.

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7, 8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H4Bip, has been attributed to enzyme-associated H4Bip. To elucidate further H4Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H4Bip and inhibited H4Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of Vmax) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H4Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H4Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H4Bip redox cycling in NOS. Rather, H4Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.

Redox properties of human medium-chain acyl-CoA dehydrogenase, modulation by charged active-site amino acid residues.

The modulation of the electron-transfer properties of human medium-chain acyl-CoA dehydrogenase (hwtMCADH) has been studied using wild-type and site-directed mutants by determining their midpoint potentials at various pH values and estimating the involved pKs. The mutants used were E376D, in which the negative charge is retained; E376Q, in which one negative charge (pKa approximately 6. 0) is removed from the active center; E99G, in which a different negative charge (pKa approximately 7.3) also is affected; and E376H (pKa approximately 9.3) in which a positive charge is present. Em for hwtMCADH at pH 7.6 is -0.114 V. Results for the site-directed mutants indicate that loss of a negative charge in the active site causes a +0.033 V potential shift. This is consistent with the assumption that electrostatic interactions (as in the case of flavodoxins) and specific charges are important in the modulation of the electron-transfer properties of this class of dehydrogenases. Specifically, these charge interactions appear to correlate with the positive Em shift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715], which coincides with a pK increase of Glu376-COOH from approximately 6 to 8-9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pKa value of approximately 6.0 is assigned to the catalytic base, Glu376-COOH, in the oxidized enzyme based on comparison with the pH behavior of the E376H mutant, it thus coincides with the pK value recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The pKa of approximately 7.1 is assigned to Glu376-COOH in reduced hwtMCADH. Comparable values for these pKas for Glu376-COOH in pig kidney MCADH are pKox = 6.5 and pKred = 7.9. The Em measured for K304E-MCADH (a major mutant resulting in a deficiency syndrome) is essentially identical to that of hwtMCADH, indicating that the disordered enzyme has an intact active site.

Activation of thiamin diphosphate in enzymes.

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.