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On The Mechanism of D-Amino Acid Oxidase

1997; Elsevier BV; Volume: 272; Issue: 8 Linguagem: Inglês

10.1074/jbc.272.8.4924

1083-351X

Loredano Pollegioni, Wolfgang Blodig, Sandro Ghisla,

Sulfur Compounds in Biology

The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [α-1H]- and [α-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of ≈8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is ≈3.9, and the solvent isotope effect ≈1.6. The reductive half-reaction is biphasic, the first, fast phase, k2, corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k−2, the reversal of k2. k2 is 28.8 and 4.6 s−1 for [α-1H]- and [α-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect ≈6. The solvent deuterium isotope effect on the apparent rate of reduction for [α-1H]- and [α-2H]phenylglycine is ≈2.8 and ≈5. The rates for k−2 are 4.2 and 0.9 s−1 for [α-1H]- and [α-2H]phenylglycine, respectively, and the corresponding isotope effect is ≈4.7. The isotope effect on α-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a symmetric transition state.The k2 and k−2 values for phenylglycines carrying the para substituents F, Cl, Br, CH3, OH, NO2 and OCH3 have been determined. There is a linear correlation of k2 with the substituent volume VM and with σ+; k−2 correlates best with σ or σ+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Brønsted plot of ΔG‡ versus ΔG0 allows the estimation of the intrinsic ΔG0‡ as ≈58 kJ·;M−1. From the linear free energy correlations, the relation of ΔG‡ versus ΔG0 and according to the theory of Marcus it is concluded that there is little if any development of charge in the transition state. This, together with the recently solved three-dimensional structure of D-amino acid oxidase from pig kidney (Mattevi, A., Vanoni, M.A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7496-7501), argues against a carbanion mechanism in its classical formulation. Our data are compatible with transfer of a hydride from the substrate αC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it. The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [α-1H]- and [α-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of ≈8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is ≈3.9, and the solvent isotope effect ≈1.6. The reductive half-reaction is biphasic, the first, fast phase, k2, corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k−2, the reversal of k2. k2 is 28.8 and 4.6 s−1 for [α-1H]- and [α-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect ≈6. The solvent deuterium isotope effect on the apparent rate of reduction for [α-1H]- and [α-2H]phenylglycine is ≈2.8 and ≈5. The rates for k−2 are 4.2 and 0.9 s−1 for [α-1H]- and [α-2H]phenylglycine, respectively, and the corresponding isotope effect is ≈4.7. The isotope effect on α-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a symmetric transition state. The k2 and k−2 values for phenylglycines carrying the para substituents F, Cl, Br, CH3, OH, NO2 and OCH3 have been determined. There is a linear correlation of k2 with the substituent volume VM and with σ+; k−2 correlates best with σ or σ+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Brønsted plot of ΔG‡ versus ΔG0 allows the estimation of the intrinsic ΔG0‡ as ≈58 kJ·;M−1. From the linear free energy correlations, the relation of ΔG‡ versus ΔG0 and according to the theory of Marcus it is concluded that there is little if any development of charge in the transition state. This, together with the recently solved three-dimensional structure of D-amino acid oxidase from pig kidney (Mattevi, A., Vanoni, M.A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7496-7501), argues against a carbanion mechanism in its classical formulation. Our data are compatible with transfer of a hydride from the substrate αC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it. INTRODUCTIOND-Amino acid oxidase (EC 1.4.3.3, DAAO) 1The abbreviations used are: DAAOD-Amino acid oxidaseAAamino acidIAimino acidTvDAAOD-amino acid oxidase from the yeast Trigonopsis variabilispkDAAOD-amino acid oxidase from pig kidneyEoxDAAO oxidized formEredDAAO reduced formEred∼IAreduced DAAO imino acid complexEox∼AAMichaelis complex between the oxidized DAAO and substrateLFERlinear free energy relationship. is the paradigm of flavin enzymes. It was the second flavoprotein to be uncovered, and probably it is the most studied member of this superfamily. In addition to the classical protein from mammalian kidney, recently related DAAOs have been described from various yeasts (1Kubicek-Pranz E.M. Röhr M. Can. J. Microbiol. 1985; 31: 625-628Google Scholar, 2Pilone Simonetta M. Vanoni M.A. Casalin P. Biochim. Biophys. Acta. 1987; 914: 136-142Google Scholar). A common feature of all these enzymes is the dehydrogenation of D-amino acids to yield α-imino and, upon subsequent hydrolysis, α-ketoacids. The terminal redox acceptor is dioxygen. In spite of the innumerable studies, the molecular mechanism by which this enzyme brings about substrate dehydrogenation is far from being solved. Mechanistic proposals revolve around possible modes by which the substrate αC-H bond is being broken in the step critical for catalysis.The most prominent proposal is the carbanion mechanism, which is characterized by initial abstraction of the α-H as H+ leading to an intermediate in which the α-carbon carries a negative charge. Evidence in its favor has been discussed in various review articles (3Bright H.J. Porter D.J.T. Enzymes. 1975; 12: 421-505Google Scholar, 4Curti B. Ronchi S. Pilone Simonetta M. Müller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Inc., Boca Raton, FL1992: 69-94Google Scholar). So called “hydride mechanisms” in which a H− is expulsed from αC-H also have been discussed at various occasions but have not been proposed explicitly until most recently by Mattevi et al. (5Mattevi A. Vanoni M.A. Todone F. Rizzi M. Teplyakov A. Coda A. Bolognesi M. Curti B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7496-7501Google Scholar). From Miura and Miyake (6Miura R. Miyake Y. Bioorg. Chem. 1988; 16: 97-110Google Scholar) stems a proposal in which “the lone-pair electrons of the neutral amino group of the substrate are transferred to the flavin in a concerted manner with the abstraction of the α-proton.” (For schematic representations of the mechanisms and structures see Denu and Fitzpatrick (7Denu J.M. Fitzpatrick P.F. Biochemistry. 1994; 33: 4001-4007Google Scholar).)An approach to investigate the molecular mechanisms of enzymes consists in the correlation of reactivities (reaction rates) with the properties of substrate substituents which influence the steric or electronic properties of the latter. This approach was advocated originally by Hammett (8Hammett L.P. Physical Organic Chemistry. McGraw-Hill Inc., New York1940: 184-228Google Scholar) for chemical systems and was extended by Hansch and Leo (9Hansch C. Leo A. Substituent Constants For Correlation Analysis in Chemistry and Biology. John Wiley & Sons, Inc., New York1979Google Scholar). Klinman and co-workers (10Klinman J.P. Biochemistry. 1976; 15: 2018-2026Google Scholar, 11Miller S.M. Klinman J.P. Biochemistry. 1985; 24: 2114-2127Google Scholar) have pioneered its use in the study of enzymatic reactions. Recently Walker and Edmondson (12Walker M.C. Edmondson D.E. Biochemistry. 1994; 33: 7088-7098Google Scholar) have used it to study monoamine oxidase. As early as in 1966 Neims et al. (13Neims A.H. DeLuca D.C. Hellerman L. Biochemistry. 1966; 5: 203-212Google Scholar) have employed substituted phenylglycines for probing the mechanism of pkDAAO; however, the results were contradictory. In retrospect the reason for this is clear: these authors did rely on the correlations of Vmax data hoping to probe the reductive half-reaction. With pkDAAO the rate-limiting step in turnover is, in general, product release (14Massey V. Gibson Q.H. Fed. Proc. Am. Chem. Soc. Exp. Biol. 1964; 23: 18-29Google Scholar). Later, Porter et al. (15Porter D.J.T. Voet J.G. Bright H.J. J. Biol. Chem. 1977; 252: 4464-4473Google Scholar) using a series of substituted phenylalanines have correlated the rate of the reversal of the dehydrogenation step of pkDAAO with the Hammett σn coefficient. They interpret their positive ρ (the numerical coefficient of σ) as compatible with a carbanion mechanism. Effects of the substituents on the rate of enzyme reduction, on ΔG0 and, by reflection of the latter on ΔG‡, did not get addressed.It has been pointed out elsewhere (16Ghisla S. Massey V. Williams C.H. Flavins and Flavoproteins. Elsevier, North-Holland, New York1982: 133-142Google Scholar–18Lederer F. Müller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Inc., Boca Raton, FL1991: 153-242Google Scholar) that the dehydrogenation of amino acids by DAAOs should be mechanistically related to that of α-OH acids as catalyzed, e.g. by lactate monooxygenase or flavocytochrome b2, to name only the two most prominent members of this family. This assumption has been substantiated nicely by Mattevi et al. (5Mattevi A. Vanoni M.A. Todone F. Rizzi M. Teplyakov A. Coda A. Bolognesi M. Curti B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7496-7501Google Scholar), who have shown that the active sites of pkDAAO and flavocytochrome b2 are mirror images resulting probably from convergent evolution. If these assumptions are correct, then the mechanistic arguments from the two subfamilies should be usable reciprocally. A major argument against a simple carbanion mechanism is, as discussed earlier (16Ghisla S. Massey V. Williams C.H. Flavins and Flavoproteins. Elsevier, North-Holland, New York1982: 133-142Google Scholar), the finding of incorporation of the substrate α-H into the 5-deazaflavin position C(5) both in the case of pkDAAO and of α-OH acid oxidases (16Ghisla S. Massey V. Williams C.H. Flavins and Flavoproteins. Elsevier, North-Holland, New York1982: 133-142Google Scholar, 19Hersh L.B. Jorns M.S. J. Biol. Chem. 1975; 250: 8728-8734Google Scholar, 20Averill B.A. Schonbrunn A. Abeles R.H. Weinstock L.T. Cheng C.C. Fisher J. Spencer R. Walsh C. J. Biol. Chem. 1975; 250: 1603-1605Google Scholar). If a carbanion mechanism was to be operative, this would require additional steps or intermediates, since the H+ originating from the αC-H cannot be transferred to the flavin N(5) or C(5) concomitantly with its abstraction, unless the flavin position N(5) (or C(5) in 5-deazaflavin) would be the “abstracting base” itself, a most unlikely alternative. From this, the importance of the question about the concertedness of the reaction, as stated and studied e.g. by Denu and Fitzpatrick (7Denu J.M. Fitzpatrick P.F. Biochemistry. 1994; 33: 4001-4007Google Scholar), becomes apparent.During our recent studies on the catalytic mechanism of the two yeast DAAOs from Rhodotorula gracilis and Trigonopsis variabilis (21Pollegioni L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Google Scholar) using aliphatic D-amino acids, major differences compared with pkDAAO have emerged. Importantly, the yeast enzymes are more tolerant of variations in the chain of the amino acid; they have, overall, much higher turnover rates and have a different rate-limiting step, e.g. with alanine as substrate it is the reductive half-reaction, compared with product release in the case of pkDAAO (14Massey V. Gibson Q.H. Fed. Proc. Am. Chem. Soc. Exp. Biol. 1964; 23: 18-29Google Scholar). In view of this we have attempted to apply the concepts of linear free energy relationships (LFER) using p-substituted phenylglycines to probe the mechanistic questions mentioned above. The rationale behind the choice of phenylglycine is that the electronic and inductive effects of substituents on the aromatic ring should be more substantial compared with those in substituted phenylalanines as studied by Porter et al. (15Porter D.J.T. Voet J.G. Bright H.J. J. Biol. Chem. 1977; 252: 4464-4473Google Scholar). We have worked out the detailed kinetic mechanism for phenylglycines since we consider this to be an indispensable basis for linear free energy interpretations. Concomitantly, we have studied the primary deuterium isotope effect (on αC-H) and the solvent deuterium isotope effect on the dehydrogenation step of phenylglycine in order to establish whether, with TvDAAO, it occurs in a concerted fashion or via intermediates. The results are interpreted in view of the recently solved three-dimensional structure of pkDAAO, the coordinates of which were made available to us prior to publication by Mattevi et al. (5Mattevi A. Vanoni M.A. Todone F. Rizzi M. Teplyakov A. Coda A. Bolognesi M. Curti B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7496-7501Google Scholar). INTRODUCTIOND-Amino acid oxidase (EC 1.4.3.3, DAAO) 1The abbreviations used are: DAAOD-Amino acid oxidaseAAamino acidIAimino acidTvDAAOD-amino acid oxidase from the yeast Trigonopsis variabilispkDAAOD-amino acid oxidase from pig kidneyEoxDAAO oxidized formEredDAAO reduced formEred∼IAreduced DAAO imino acid complexEox∼AAMichaelis complex between the oxidized DAAO and substrateLFERlinear free energy relationship. is the paradigm of flavin enzymes. It was the second flavoprotein to be uncovered, and probably it is the most studied member of this superfamily. In addition to the classical protein from mammalian kidney, recently related DAAOs have been described from various yeasts (1Kubicek-Pranz E.M. Röhr M. Can. J. Microbiol. 1985; 31: 625-628Google Scholar, 2Pilone Simonetta M. Vanoni M.A. Casalin P. Biochim. Biophys. Acta. 1987; 914: 136-142Google Scholar). A common feature of all these enzymes is the dehydrogenation of D-amino acids to yield α-imino and, upon subsequent hydrolysis, α-ketoacids. The terminal redox acceptor is dioxygen. In spite of the innumerable studies, the molecular mechanism by which this enzyme brings about substrate dehydrogenation is far from being solved. Mechanistic proposals revolve around possible modes by which the substrate αC-H bond is being broken in the step critical for catalysis.The most prominent proposal is the carbanion mechanism, which is characterized by initial abstraction of the α-H as H+ leading to an intermediate in which the α-carbon carries a negative charge. Evidence in its favor has been discussed in various review articles (3Bright H.J. Porter D.J.T. Enzymes. 1975; 12: 421-505Google Scholar, 4Curti B. Ronchi S. Pilone Simonetta M. Müller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Inc., Boca Raton, FL1992: 69-94Google Scholar). So called “hydride mechanisms” in which a H− is expulsed from αC-H also have been discussed at various occasions but have not been proposed explicitly until most recently by Mattevi et al. (5Mattevi A. Vanoni M.A. Todone F. Rizzi M. Teplyakov A. Coda A. Bolognesi M. Curti B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7496-7501Google Scholar). From Miura and Miyake (6Miura R. Miyake Y. Bioorg. Chem. 1988; 16: 97-110Google Scholar) stems a proposal in which “the lone-pair electrons of the neutral amino group of the substrate are transferred to the flavin in a concerted manner with the abstraction of the α-proton.” (For schematic representations of the mechanisms and structures see Denu and Fitzpatrick (7Denu J.M. Fitzpatrick P.F. Biochemistry. 1994; 33: 4001-4007Google Scholar).)An approach to investigate the molecular mechanisms of enzymes consists in the correlation of reactivities (reaction rates) with the properties of substrate substituents which influence the steric or electronic properties of the latter. This approach was advocated originally by Hammett (8Hammett L.P. Physical Organic Chemistry. McGraw-Hill Inc., New York1940: 184-228Google Scholar) for chemical systems and was extended by Hansch and Leo (9Hansch C. Leo A. Substituent Constants For Correlation Analysis in Chemistry and Biology. John Wiley & Sons, Inc., New York1979Google Scholar). Klinman and co-workers (10Klinman J.P. Biochemistry. 1976; 15: 2018-2026Google Scholar, 11Miller S.M. Klinman J.P. Biochemistry. 1985; 24: 2114-2127Google Scholar) have pioneered its use in the study of enzymatic reactions. Recently Walker and Edmondson (12Walker M.C. Edmondson D.E. Biochemistry. 1994; 33: 7088-7098Google Scholar) have used it to study monoamine oxidase. As early as in 1966 Neims et al. (13Neims A.H. DeLuca D.C. Hellerman L. Biochemistry. 1966; 5: 203-212Google Scholar) have employed substituted phenylglycines for probing the mechanism of pkDAAO; however, the results were contradictory. In retrospect the reason for this is clear: these authors did rely on the correlations of Vmax data hoping to probe the reductive half-reaction. With pkDAAO the rate-limiting step in turnover is, in general, product release (14Massey V. Gibson Q.H. Fed. Proc. Am. Chem. Soc. Exp. Biol. 1964; 23: 18-29Google Scholar). Later, Porter et al. (15Porter D.J.T. Voet J.G. Bright H.J. J. Biol. Chem. 1977; 252: 4464-4473Google Scholar) using a series of substituted phenylalanines have correlated the rate of the reversal of the dehydrogenation step of pkDAAO with the Hammett σn coefficient. They interpret their positive ρ (the numerical coefficient of σ) as compatible with a carbanion mechanism. Effects of the substituents on the rate of enzyme reduction, on ΔG0 and, by reflection of the latter on ΔG‡, did not get addressed.It has been pointed out elsewhere (16Ghisla S. Massey V. Williams C.H. Flavins and Flavoproteins. Elsevier, North-Holland, New York1982: 133-142Google Scholar–18Lederer F. Müller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Inc., Boca Raton, FL1991: 153-242Google Scholar) that the dehydrogenation of amino acids by DAAOs should be mechanistically related to that of α-OH acids as catalyzed, e.g. by lactate monooxygenase or flavocytochrome b2, to name only the two most prominent members of this family. This assumption has been substantiated nicely by Mattevi et al. (5Mattevi A. Vanoni M.A. Todone F. Rizzi M. Teplyakov A. Coda A. Bolognesi M. Curti B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7496-7501Google Scholar), who have shown that the active sites of pkDAAO and flavocytochrome b2 are mirror images resulting probably from convergent evolution. If these assumptions are correct, then the mechanistic arguments from the two subfamilies should be usable reciprocally. A major argument against a simple carbanion mechanism is, as discussed earlier (16Ghisla S. Massey V. Williams C.H. Flavins and Flavoproteins. Elsevier, North-Holland, New York1982: 133-142Google Scholar), the finding of incorporation of the substrate α-H into the 5-deazaflavin position C(5) both in the case of pkDAAO and of α-OH acid oxidases (16Ghisla S. Massey V. Williams C.H. Flavins and Flavoproteins. Elsevier, North-Holland, New York1982: 133-142Google Scholar, 19Hersh L.B. Jorns M.S. J. Biol. Chem. 1975; 250: 8728-8734Google Scholar, 20Averill B.A. Schonbrunn A. Abeles R.H. Weinstock L.T. Cheng C.C. Fisher J. Spencer R. Walsh C. J. Biol. Chem. 1975; 250: 1603-1605Google Scholar). If a carbanion mechanism was to be operative, this would require additional steps or intermediates, since the H+ originating from the αC-H cannot be transferred to the flavin N(5) or C(5) concomitantly with its abstraction, unless the flavin position N(5) (or C(5) in 5-deazaflavin) would be the “abstracting base” itself, a most unlikely alternative. From this, the importance of the question about the concertedness of the reaction, as stated and studied e.g. by Denu and Fitzpatrick (7Denu J.M. Fitzpatrick P.F. Biochemistry. 1994; 33: 4001-4007Google Scholar), becomes apparent.During our recent studies on the catalytic mechanism of the two yeast DAAOs from Rhodotorula gracilis and Trigonopsis variabilis (21Pollegioni L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Google Scholar) using aliphatic D-amino acids, major differences compared with pkDAAO have emerged. Importantly, the yeast enzymes are more tolerant of variations in the chain of the amino acid; they have, overall, much higher turnover rates and have a different rate-limiting step, e.g. with alanine as substrate it is the reductive half-reaction, compared with product release in the case of pkDAAO (14Massey V. Gibson Q.H. Fed. Proc. Am. Chem. Soc. Exp. Biol. 1964; 23: 18-29Google Scholar). In view of this we have attempted to apply the concepts of linear free energy relationships (LFER) using p-substituted phenylglycines to probe the mechanistic questions mentioned above. The rationale behind the choice of phenylglycine is that the electronic and inductive effects of substituents on the aromatic ring should be more substantial compared with those in substituted phenylalanines as studied by Porter et al. (15Porter D.J.T. Voet J.G. Bright H.J. J. Biol. Chem. 1977; 252: 4464-4473Google Scholar). We have worked out the detailed kinetic mechanism for phenylglycines since we consider this to be an indispensable basis for linear free energy interpretations. Concomitantly, we have studied the primary deuterium isotope effect (on αC-H) and the solvent deuterium isotope effect on the dehydrogenation step of phenylglycine in order to establish whether, with TvDAAO, it occurs in a concerted fashion or via intermediates. The results are interpreted in view of the recently solved three-dimensional structure of pkDAAO, the coordinates of which were made available to us prior to publication by Mattevi et al. (5Mattevi A. Vanoni M.A. Todone F. Rizzi M. Teplyakov A. Coda A. Bolognesi M. Curti B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7496-7501Google Scholar).