ON THE MECHANISM OF D-AMINO-ACID OXIDASE - STRUCTURE LINEAR FREE-ENERGY CORRELATIONS AND DEUTERIUM KINETIC ISOTOPE EFFECTS USING SUBSTITUTED PHENYLGLYCINES/

The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4. 3.3) from Trigonopsis variabilis with [alpha-H-1]- and [alpha-H-2]phen ylglycine has been determined. The pH dependence of V-max is compatibl e with pK(a) values of approximate to 8.1 and >9.5, the former of whic h is attributed to a base which should be deprotonated for efficient c atalysis. The deuterium isotope effect on turnover is approximate to 3 .9, and the solvent isotope effect approximate to 1.6. The reductive h alf-reaction is biphasic, the first, fast phase, k(2), corresponding t o 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 reversa l of k(2). k(2) is 28.8 and 4.6 s(-1) for [alpha-H-1]- and [alpha-H-2] phenylglycine, respectively, yielding a primary deuterium iso tope eff ect approximate to 6. The solvent deuterium isotope effect on the appa rent rate of reduction for [alpha-H-1]- and [alpha-H-2]phenylglycine i s approximate to 2.8 and approximate to 5. The rates for k(-2), are 4. 2 and 0.9 s(-1) for [alpha-H-1]- and [alpha-H-2]phenylglycine, respect ively, and the corresponding isotope effect is approximate to 4.7. The isotope effect on alpha-H and the solvent one thus behave multiplicat ively consistent with a highly concerted process and a symmetric trans ition state. The k(2) and k(-2) values for phenylglycines carrying the para substituents F, Cl, Br, CH3, OH, NO2 and OCH3 have been determin ed. There is a Linear correlation of k(2) with the substituent volume V-M and with sigma(+); k(-2) correlates best with sigma or sigma(+) wh ile steric parameters have little influence. This is consistent with t he transition state being structurally similar to the product. The Bro nsted plot of Delta G(double dagger) versus Delta G(0) allows the esti mation of the intrinsic Delta G(0)(double dagger) as approximate to 58 kJ . M(-1). From the linear free energy correlations, the relation of Delta G(double dagger) versus Delta G(0) and according to the theory of Marcus it is concluded that there is little if any development of c harge 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 classic al formulation. Our data are compatible with transfer of a hydride fro m the substrate alpha C-H to the oxidized flavin N(5) position, althou gh, clearly, they cannot prove it.