On The Mechanism of D-Amino Acid Oxidase: STRUCTURE/LINEAR FREE ENERGY CORRELATIONS AND DEUTERIUM KINETIC ISOTOPE EFFECTS USING SUBSTITUTED PHENYLGLYCINES

Abstract

The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [alpha-1H]- and [alpha-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of approximately 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 approximately 3.9, and the solvent isotope effect approximately 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 [alpha-1H]- and [alpha-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect approximately 6. The solvent deuterium isotope effect on the apparent rate of reduction for [alpha-1H]- and [alpha-2H]phenylglycine is approximately 2.8 and approximately 5. The rates for k-2 are 4.2 and 0.9 s-1 for [alpha-1H]- and [alpha-2H]phenylglycine, respectively, and the corresponding isotope effect is approximately 4.7. The isotope effect on alpha-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 sigma+; k-2 correlates best with sigma or sigma+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Bronsted plot of DeltaG versus DeltaG0 allows the estimation of the intrinsic DeltaG0 as approximately 58 kJ.M-1. From the linear free energy correlations, the relation of DeltaG versus DeltaG0 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 alphaC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it.