On The Mechanism of D-Amino Acid Oxidase

Abstract

The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1. 4.3.3) from Trigonopsis variabilis with [␣-1 H]-and [␣-2 H]phenylglycine has been determined. The pH dependence of V max is compatible with pK a values of Ϸ8.1 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 Ϸ3.9, and the solvent isotope effect Ϸ1.6. The reductive halfreaction is biphasic, the first, fast phase, k 2 , 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 k 2 . D-Amino acid oxidase (EC 1.4.3.3, DAAO) 1 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 (1, 2). A common feature of all these enzymes is the dehydrogenation of D-amino acids to yield ␣-imino and, upon subsequent hydrolysis, ␣-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 ␣C-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 ␣-H as H ϩ leading to an intermediate in which the ␣-carbon carries a negative charge. Evidence in its favor has been discussed in various review articles (3, 4). So called "hydride mechanisms" in which a H Ϫ is expulsed from ␣C-H also have been discussed at various occasions but have not been proposed explicitly until most recently by Mattevi et al. (5). From Miura and Miyake (6) 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 ␣-proton." (For schematic representations of the mechanisms and structures see Denu and Fitzpatrick (7).) 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 (8) for chemical systems and was extended by Hansch and Leo (9). Klinman and co-workers (10, 11) have pioneered its use in the study of enzymatic reactions. Recently Walker and Edmondson (12) have used it to study monoamine oxidase. As early as in 1966 Neims et al. (13) 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 V max da...

Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

“…The C2 position of GAP is in proximity to N5 of the flavin at 4.2 Å; this distance suggests complexation with the endogenous substrate, G3P, likely induces a conformational shift of Ϸ1 Å so that the transition state distances would be more optimal for direct hydride transfer. The proposed direct hydride transfer mechanism is consistent with those elaborated for D-amino acid oxidase (25), PutA669 (26), and complex II (27). The reduced flavin is stabilized by Lys-354 (Fig.…”

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

“…On the other hand, Mattevi's group favored a classical hydride mechanism based on the mode of interaction of substrate (D-alanine) modeled into the active site of pkDAAO (7). This latter hypothesis is supported by linear free energy correlations, which indicate that no significant charge develops in the transition state of the reaction catalyzed by Trigonopsis variabilis DAAO (11). Kinetic isotope effects are consistent with a concerted cleavage of the substrate COH and NOH bonds in the catalytic reaction and argue against the occurrence of intermediates (11).…”

Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase

“…The reversible reduction of the flavin in D170E is followed by a substrate concentration-dependent second step in the reductive half-reaction, which reflects the decomposition of the p-quinone methide intermediate-reduced enzyme complex (k 3 ). This substrate-dependent phenomenon was also reported for D-amino acid oxidase in case of k -2 being an important term in the reductive half-reaction (31,32).…”