The influence of para and meta substitution of benzylamine on its interaction with bovine liver mitochondrial monoamine oxidase B (MAO B) has been investigated by steady-state and reductive half-reaction anaerobic stopped-flow kinetic approaches. Steady-state kinetic properties of each benzylamine analogue suggest that para or meta substitution does not-alter the mechanistic pathway of catalysis [Husain, M., et al. (1982) Biochemistry 21, 595-600]. All analogues tested exhibited (D)k(cat) values ranging from 5.5 to 8.9 and (D)[k(cat)/K-m(amine)] values ranging from 3.3 to 8.1. (D)[k(cat)/K-m(O-2)] values of similar to 1 are observed for all substrate analogues. Values for K-d were calculated from steady-state isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Sec. 107, 1058-1060] and are in good agreement with K-s values determined from analysis of the rate of MAO B reduction as a function of benzylamine analogue concentration in reductive half-reaction experiments. A linear correlation of benzylamine analogue K-d values with the hydrophobicity parameter (pi) is observed for the para-substituted analogues where the binding affinity increases with increasing hydrophobicity of the substituent. Statistical treatment of the correlation shows a small negative contribution to binding by the van der Waals volume (V-W) of the para substituent. meta-Substituted benzylamine analogues show a decreased binding affinity with the V-W of the substituent and no correlation with the hydrophobicity value of the substituents tested. No spectral evidence was found for any flavin radical intermediates during the time course of MAO B flavin reduction in anaerobic reductive half-reduction stopped-flow experiments with any of the alpha,alpha-diprotio- or alpha,alpha-dideuteriobenzylamine analogues tested. The limiting rates of enzyme reduction exhibit large (D)k values (6.5-14.1) for all of the analogues tested. para-Substituted benzylamine analogues reduce MAO B with limiting rates that correlate with the steric influence (E(s) value) of the substituent. Statistical analysis shows the rate of MAO B reduction by para-substituted analogues to be retarded by increased values of E(s) and, with a smaller contribution, by the hydrophobicity value of the substituent. The rate of MAO B reduction by meta-substituted benzylamine analogues is essentially independent of the nature of the substituent. No evidence was found for any electronic contribution to the rate of MAO B flavin reduction by any of the analogues tested. These data demonstrate the steric orientation of the substrate to be important in the rate of amine oxidation by MAO B and that ring meta substituents favor this orientation. The relevance of these results to the proposed radical mechanism for MAO catalysis [Silverman, R. B. (1991) Biochem. Sec. Trans. 19, 201-206] is discussed.
