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V Massey

Publications and source records attributed to V Massey.

At least 73 records · Page 4Linked to original sources
PubMed1995

Old Yellow enzyme: aromatization of cyclic enones and the mechanism of a novel dismutation reaction.

The origin of charge transfer bands that develop on reaction of Old Yellow Enzyme with alpha,beta-unsaturated cyclic ketones such as 3-oxodecalin-4-ene (ODE, numbered according to the convention for steroids), 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC), and 2-cyclohexenone is shown to be due to the aromatization of ODE and ODEC to 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and of 2-cyclohexenone to phenol. The aromatization of ODEC to HTN is stereospecific and involves the trans dehydrogenation of the 1 beta, 2 alpha hydrogens. The aromatization occurs under aerobic as well as anaerobic conditions. With the exception of ODEC under aerobic conditions, the aromatization of these substrates is accompanied by a dismutation reaction in which the olefinic bond of a second molecule of each substrate is reduced to give the saturated cyclic ketone. Molecular oxygen may serve as the electron acceptor with ODEC and some other substrates under aerobic reaction conditions. The dismutation reaction involves an overall sequence of hydride transfer from one substrate molecule to the beta-carbon of a second substrate molecule along with a solvent proton uptake by the alpha-carbon. 19-Nortestosterone is aromatized to beta-estradiol; however, other 3-oxo-delta 4-steroids such as progesterone, testosterone, and androstene-3,17-dione bind tightly to the enzyme but are not aromatized. The NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds is limited to aldehydes and ketones. alpha,beta-Unsaturated acids, esters, amides, and nitriles are not reduced. The reduction of the olefinic bond of ODE or cinnamaldehyde by NADPH occurs by an overall sequence of hydride transfer from the reduced pyridine nucleotide to the beta-carbon of the alpha,beta-unsaturated carbonyl compound and a solvent proton uptake by the alpha-carbon. The 4-pro-R hydride of NADPH is transferred in the reduction reaction. Structure-function relationships in the NADPH-dependent reduction of alpha,beta-unsaturated aldehydes or ketones indicate that increasing alkyl substitution at the beta-carbon results in marked decrease in the rate of reduction of the olefinic bond, consistent with a steric hindrance to hydride transfer at the beta-carbon.

Acrolein↗
PubMed1995

Active site studies of DT-diaphorase employing artificial flavins.

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.

Binding Sites↗
PubMed1995

A new old yellow enzyme of Saccharomyces cerevisiae.

In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D. J., and Massey, V. (1993) J. Biol. Chem. 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). The analysis of an OYE2 deletion mutant suggested that S. cerevisiae had at least two OYE genes. In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli. OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence. In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence. Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined. Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.

Amino Acid Sequence↗
PubMed1995

DT-diaphorase. Redox potential, steady-state, and rapid reaction studies.

NAD(P)H:quinone oxidoreductase (DT-diaphorse) appears to be a 2-electron transfer flavoprotein, which catalyzes the conversion of quinones into hydroquinones. Upon photoreduction in the presence of dimethylformamide, the enzyme forms a red semiquinone. In the absence of dimethylformamide, only 10% of the radical form is thermodynamically stabilized. This indicates a redox potential of the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple. The 2-electron redox potential was determined to be -159 +/- 3 mV at 25 degrees C, pH 7.0. In the presence of benzoquinone or 3-aminopyridine adenine dinucleotide phosphate, as NADPH analogue, there is no change in the redox properties of the enzyme flavin. A significant decrease is observed in the presence of the competitive inhibitor dicumarol (Em = -234 +/- 2 mV at pH 7.0). The reaction mechanism of the flavoprotein has been investigated by steady-state and stopped-flow kinetic methods using NADPH, NADH, deamino-NADPH, and 3-acetylpyridine adenine dinucleotide reduced form (APADH) as electron donors and K3Fe(CN)6, 4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ), and 2,5-diaziridinyl-3,6-bis(carboethoxy-amino)-1,4-benzoquinone (AZQ) as electron acceptors in 50 mM phosphate buffer, pH 7.0, 25 degrees C. No evidence could be obtained to indicate that semiquinoid intermediates play a part in the catalytic mechanism of DT-diaphorase with quinones as acceptors. The rates of the reduction by NADPH, NADH, deamino-NADPH, and APADH (1.3 x 10(9), 8.8 x 10(8), 8.3 x 10(8) and 9.8 x 10(8) m-1 min-1, respectively) as well as the rates of the reoxidation by PQQ and AZQ (9 x 10(4) and 2.8 x 10(6) M-1 min-1, respectively) are directly proportional to substrate concentration, and there is no evidence of the formation of enzyme-substrate complexes. If such complexes do indeed exist, the affinity of the enzyme for substrate must be extremely low. Using K3Fe(CN)6 as electron acceptor, the rate of oxidation of fully reduced enzyme is 4.6 x 10(7) M-1 min-1 and it is accurately proportional to ferricyanide concentration. This rate represents that of flavin semiquinone formation, with the subsequent oxidation of the semiquinone being much faster, since no spectral evidence for semiquinone formation could be obtained. Studies were also conducted attempting to use apo-DT-diaphorase reconstituted with PQQ as coenzyme. The lack of activity toward AZQ, K3Fe(CN)6, and menadione suggests that DT-diaphorase can use PQQ only as electron acceptor and not as redox cofactor.

Flavin-Adenine Dinucleotide↗
PubMed1995

L-lactate oxidase and L-lactate monooxygenase: mechanistic variations on a common structural theme.

Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-alpha-hydroxy acids, including flavocytochrome b2, lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydroxy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many properties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable and is the form that reacts with O2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O2, to give the observed products, pyruvate and H2O2.

Amino Acid Sequence↗
PubMed1995

Flavoprotein structure and mechanism. 8. Structure-function relations for old yellow enzyme.

The past 5 years have seen tremendous progress in our knowledge of old yellow enzyme (OYE) as a number of OYEs have been cloned and expressed, a high-resolution crystal structure has been determined for one of these, and new substrates have been found that can be turned over by the enzyme. Together these studies do not yet define the physiological role of OYE, but they lead to significant new insights into the enzymatic properties and structure-function relations of OYE.

Amino Acid Sequence↗
PubMed1994

Studies on the kinetic mechanism of pig kidney D-amino acid oxidase by site-directed mutagenesis of tyrosine 224 and tyrosine 228.

Expression conditions in Escherichia coli of wild-type, Y224F, and Y228F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties similar to those of the wild-type enzyme, in all oxidation-reduction states. All enzymes were studied by steady state and rapid reaction methods. Turnover numbers determined for Y224F DAAO with different substrates were similar to those of wild-type protein, while the Y228F DAAO always showed lower turnover numbers and higher Km values for the D-amino acid. Analyses of reduction traces at 450 and 550 nm of stopped-flow experiments with wild-type DAAO showed the presence of a new phase, the conversion between two different charge-transfer complexes of the reduced enzyme and imino acid product. The substitution of Tyr-228 totally abolished the formation of the long wavelength bands while Y224F DAAO showed long wavelength absorbance only for the first intermediate. Reoxidation of the reduced flavin results from reaction of oxygen with the first charge-transfer complex. The rate of reduction with D-alanine as substrate was 1225,45 and 10 s-1 for wild-type, Y224F, and Y228F DAAOs, respectively. Comparison of the properties of these two mutant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-reaction of the enzyme.

Animals↗
PubMed1994

The mobile flavin of 4-OH benzoate hydroxylase.

Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

Benzoate 4-Monooxygenase↗
PubMed1994

Studies of the reductive half-reaction of milk xanthine dehydrogenase.

The reductive half-reaction of milk xanthine dehydrogenase (XDH) with NADH and with xanthine has been studied at pH 7.5, 25 degree C. NADH reduces XDH to the two-electron reduced form at a rate of 18 s-1, independent of NADH concentration over the range studied. Further reduction by NADH to the four-electron state is inhibited by excess NADH. Subsequent binding of NADH to the four-electron reduced form of the enzyme causes the redistribution of one electron from the flavin to the molybdenum center. The four-electron reduced species reached through reduction by NADH is the same as the species obtained upon reaction of NAD with fully reduced XDH. In contrast, xanthine rapidly reduces XDH to the four-electron level; further reduction is comparatively slow and is inhibited by excess xanthine. Studies using substoichiometric xanthine show that the reaction of XDH with 1 equivalent of xanthine involves rapid substrate binding and rapid reduction of the molybdenum center of the enzyme. Before the release of urate from the molybdenum active site, an electron is transferred at 15 s-1 from the reduced molybdenum center to one of the iron-sulfur centers of XDH. Urate is then released at a rate of 13 s-1, followed by a rapid electron redistribution within the protein. The reductive half-reaction of XDH with xanthine is rate-limiting in xanthine/NAD turnover, which appears to occur between the two- and four-electron reduced enzyme species. The reduction of XDH by substoichiometric amounts of the fluorescent substrate xanthopterin was also studied. This reaction, monitored by changes in both absorbance and fluorescence, was found to involve the formation of two molybdenum complexes (an Eox.S complex and an Ered.P complex) followed by the release of the product, leucopterin.

Animals↗
PubMed1994

Modulation of the oxidation-reduction potential of the flavin in lipoamide dehydrogenase from Escherichia coli by alteration of a nearby charged residue, K53R.

The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD [Pai & Schulz (1983) J. Biol. Chem. 258, 1752]. A conserved lysine residue occupies a similar position in lipoamide dehydrogenase. This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R. The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD+ are profoundly affected by the change. K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate. The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pKa = 7.0. Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum and indicates an apparent Kd = 7.0 microM at pH 7.6. The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD+. K53R is extensively reduced (mostly EH4) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH2). The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1994

Studies on the oxidative half-reaction of p-hydroxyphenylacetate 3-hydroxylase.

The oxidative half-reaction of the two-protein enzyme, p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, has been studied by absorbance stopped-flow techniques. The formation of three flavin-oxygen intermediates, the anionic and protonated forms of the flavin hydroperoxide (intermediates I and I) and the hydroxyflavin (intermediate III), was observed during the course of the oxygen reaction with the reduced flavoprotein-coupling protein complex. The flavin hydroperoxide, which is formed in a second-order reaction with oxygen, is in rapid equilibrium with the aromatic substrate, p-hydroxyphenylacetate. Due to this rapid equilibrium, p-hydroxyphenylacetate effectively competes with other ligands, such as p-chlorophenylacetate and p-aminophenylacetate and proceeds through the hydroxylation pathway. Furthermore, dehydration of intermediate III is subjected to severe inhibition in the presence of excess p-hydroxyphenylacetate, similar to the observations made with phenol hydroxylase. A reaction mechanism for the oxidative half-reaction in the presence of the aromatic substrate, p-hydroxyphenylacetate, is proposed.

Kinetics↗
PubMed1994

Lactate monooxygenase. I. Expression of the mycobacterial gene in Escherichia coli and site-directed mutagenesis of lysine 266.

Lactate monooxygenase utilizes oxygen in the conversion of L-lactate to acetate, CO2, and water. The gene for the enzyme from Mycobacterium smegmatis had been cloned into Escherichia coli (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and the derived amino acid sequence compared to glycolate oxidase and flavocytochrome b2, enzymes of known three-dimensional structure (Lindqvist, Y., and Brändén, C. I. (1989) J. Biol. Chem. 264, 3624-3628; Xia, Z. X., and Mathews, S. F. (1990) J. Mol. Biol. 212, 837-863). There is strong homology, especially around residues in the active site. The mechanism proposed for lactate monooxygenase involves an intermediate having a negative charge at the N(1)-position of the FMN. Based on the homology, lysine 266 is the residue suggested to neutralize that charge. Wild type enzyme and several forms of the enzyme altered at active site residues by site-directed mutagenesis have been expressed in E. coli and purification procedures developed. The properties determined for the recombinant wild type enzyme were, in every case, the same as those previously determined for the enzyme isolated from M. smegmatis. Mutation of lysine 266 to a methionine created K266M. The semiquinone showed spectral features different from those found in the wild type enzyme and was no longer thermodynamically stable. This indicates a redox potential for the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple. The two-electron redox potential was determined to be -180 mV at 25 degrees C, pH 7.0. In wild type enzyme, attack of the flavin ring by sulfite creates a negative charge at the FMN N(1)-position. In K266M, the stabilization of the sulfite adduct was 17,000-fold weaker (Kd approximately 10(-3) M) than in the wild type enzyme, with a rate of association that is lowered by 10,000-fold (kon = 1.2 M-1 s-1). The rate of reduction with L-lactate is significantly decreased in K266M. Unexpectedly, binding of substrate and inhibitors is significantly weaker in K266M than in the wild type enzyme. In all properties involving a negative charge at position N(1) of the FMN, K266M is distinctly different from wild type enzyme. This makes it quite likely that lysine 266 serves the postulated role of interacting with this negative charge.

Amino Acid Sequence↗
PubMed1994

Lactate monooxygenase. II. Site-directed mutagenesis of the postulated active site base histidine 290.

Lactate monooxygenase catalyzes the oxidation of L-lactate with molecular oxygen to acetate, CO2, and water. Histidine 290 has been proposed to be the active site base in lactate monooxygenase (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and was mutated to a glutamine (H290Q). The mutant enzyme shows properties that support strongly the postulated function of the histidine. The ability of L-lactate to reduce the enzyme flavin is essentially abolished, whereas reoxidation of reduced enzyme with oxygen proceeds at 1.4 x 10(4) M-1 s-1, a rate essentially like that found in the wild type enzyme. The substrate, L-lactate, is bound with a Kd equal to 2.0 x 10(-2) M, and D-lactate, a competitive inhibitor with a Kd of 3.1 x 10(-3) M. Both values are similar to binding measured in the wild type enzyme. Unlike the situation with wild type enzyme, where the transition state analog oxalate is bound tightly in a two-step reaction involving proton uptake from solution (Ghisla, S., and Massey, V. (1977) J. Biol. Chem. 252, 6729-6735), the mutant enzyme binds oxalate weakly, in a single step reaction, with a Kd in the order of 0.1 M. No effect was observed upon varying the pH, indicating that binding does not include a protonation step. Replacing the histidine also has a significant effect on the ability of the enzyme to stabilize the flavin N(5)-sulfite adduct. Sulfite is bound at least 1000-fold weaker than it is in the wild type enzyme.

Amino Acid Sequence↗
PubMed1994

Lactate monooxygenase. III. Additive contributions of active site residues to catalytic efficiency and stabilization of an anionic transition state.

Lactate monooxygenase catalyzes the conversion of L-lactate to acetate, CO2, and water with incorporation of molecular oxygen. Several amino acid residues of lactate monooxygenase had been postulated to interact in specific ways with the bound substrate (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632). Tyrosine 44 and arginine 293 were proposed to form a hydrogen bond and a salt bridge to the carboxyl-moiety of lactate. Tyrosine 152 was suggested to form a hydrogen bond to the alpha-hydroxyl group and could be involved in stabilizing a transient carbanionic intermediate of the substrate. The tyrosine residues were replaced with phenylalanines (Y44F, Y152F), and arginine 293 was mutated to a lysine (R293K). In all cases catalysis was significantly decreased; however, the binding affinity for L-lactate did not decrease. Instead, the Kd measured for Y152F was 10-fold lower than that for the wild type enzyme. The products of turnover with Y152F were similar to those with wild type enzyme, with 70-80% of the reaction proceeding to form acetate, CO2, and H2O. The catalytic reactions with both Y44F and R293K were substantially uncoupled, with between 60 and 80% of the catalytic turnover forming pyruvate and H2O2. For all mutant forms the reoxidation of enzyme with oxygen in the absence of pyruvate occurred at a rate similar to that measured for the wild type enzyme. The most important effect of the mutations was in the ability to stabilize the transition state analog oxalate. A linear relationship was found between the rate of reduction of the enzyme flavin and the dissociation constant for the binding of oxalate, demonstrating that many individual residues contribute to the lowering of the energy of the transition state, in addition to specific functions being assignable to some specific residues.

Amino Acid Sequence↗
PubMed1994

Changes in the catalytic properties of p-hydroxybenzoate hydroxylase caused by the mutation Asn300Asp.

By site-directed mutagenesis, we have changed Asn300 to Asp in p-hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) from Pseudomonas aeruginosa. In the wild-type (WT) enzyme, residue 300 is in contact with the isoalloxazine ring of the active-site FAD; in the Asn300Asp mutant, this side chain has moved by about 5 A, altering the protein structure [Lah, M.S., Palfey, B.A., Schreuder, H.A., & Ludwig, M.L. (1994) Biochemistry (following paper in this issue)]. The structural changes are responsible for profound catalytic and dynamic effects. The flavin of PHBH is reduced by NADPH in the first half of catalysis. The mutation has decreased this rate 330-fold, apparently by affecting the reactive orientation of the isoalloxazine and pyridine rings. Furthermore, the redox potential of the flavin is lower in the mutant enzyme than in WT by 20-40 mV. The reduced flavin of PHBH reacts with O2 to form a flavin C(4a)-hydroperoxide, which is the species that transfers oxygen to the aromatic substrate. Previous studies indicated that the enzyme promotes the hydroxylation reaction in part by activating the substrate through lowering the phenolic pKa. The Asn300Asp mutant does not lower the substrate pKa. As a consequence of this, and also an enhanced stability of the flavin C(4a)-hydroperoxide, the hydroxylation is 50-fold slower in the mutant than in WT. However, despite the slow rate of the hydroxylation reaction, no H2O2 is formed by the competitive elimination reaction. The kinetic stability of the flavin C(4a)-hydroxide formed by the hydroxylation was also enhanced by the mutation. By studying the effects of the inhibitor azide on the oxidative sequence, we were able to conclude that the inhibitory site is readily accessible to solvent; azide binding at a second site slowly displaces the substrate from the reduced enzyme. The mutation has profoundly slowed the rates of ligand binding to the enzyme. Kinetic studies of binding indicated the presence of several enzyme conformations. Thus, the mutation of this one residue interferes with the orientation of pyridine nucleotide and flavin during reduction, stabilizes flavin C(4a) intermediates, prevents substrate ionization, and alters the rates and strengths of ligand binding.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1994

Mechanism of p-hydroxyphenylacetate-3-hydroxylase. A two-protein enzyme.

p-Hydroxyphenylacetate-3-hydroxylase purified from Pseudomonas putida is a two-protein enzyme requiring a flavoprotein and a coupling protein for productive hydroxylation (Arunachalam, U., Massey, V., and Vaidyanathan, C. S. (1992) J. Biol. Chem. 267, 25848-25855). This paper presents information on the mechanism of the enzyme from absorbance and fluorescence stopped-flow studies. The reduction of the substrate-free flavoprotein by NADH was slow and was not altered by the presence of the coupling protein. In contrast, the coupling protein has a dramatic effect in the oxidative half-reaction. The flavoprotein when present alone, both in the absence and presence of the aromatic substrate, reacts in a second-order fashion with oxygen to form oxidized flavoprotein, with no indication of flavin-oxygen intermediates. However, an intermediate identified as the C4a-flavin hydroperoxide is stabilized when the flavoprotein-coupling protein complex reacts with oxygen in the absence of the aromatic substrate, p-hydroxyphenylacetate, and at least three flavin-oxygen intermediates, attributed to the anionic (I) and protonated (I*) forms of the flavin hydroperoxide and the hydroxyflavin (III), are formed in the oxidative half-reaction in the presence of the aromatic substrate. A reaction mechanism for the two-protein complex is proposed in which the aromatic substrate has little effect on the rate of reduction of the enzyme flavin but has strict control in the oxidative half-reaction. In this phase the flavin hydroperoxide is remarkably stable in the absence of the substrate but disappears rapidly upon encountering the aromatic substrate.

Azides↗