STRUCTURE-FUNCTION STUDIES OF SUBSTRATE OXIDATION BY BOVINE SERUM AMINE OXIDASE - RELATIONSHIP TO COFACTOR STRUCTURE AND MECHANISM

The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on k(cat) and k(cat)/K(m) parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (K(d)). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects. Multiple regression analysis yielded an electronic effect of rho = 1.47 +/- 0.27 for the bond cleavage step, supporting the intermediacy of a carbanion species. An additional effect, determined from regression analysis, indicated inhibition of catalysis by hydrophobic substituents (pi = -0.71 +/- 0.21). These results lead to a reaction mechanism for amine oxidation by the covalently bound cofactor in bovine serum amine oxidase, 6-hydroxydopa [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., & Klinman, J. P. (1990) Science 248, 981].