Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction.

Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as two half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by dioxygen. Anaerobic stopped-flow kinetic measurements of the first half-reaction indicate very large deuterium isotope effects for benzylamine, m-tyramine, and dopamine, Dk = 13.5 +/- 1.3, which are ascribed to an intrinsic isotope effect. From the insensitivity of these isotope effects to amine concentration, stopped-flow data provide substrate dissociation constants, K1, and rate constants for the C-H bond cleavage step, k3, directly. Steady-state isotope effects have also been measured for benzylamine and six ring-substituted phenethylamines. Whereas a small range of values for kcat, 0.38-1.2 s-1, and Dkcat, 5.4-8.8, is observed, kcat/Km = 1.3 X 10(2) to 3.8 X 10(4) M-1 S-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier [Miller, S., & Klinman, J.P. (1982) Methods Enzymol. 87, 711], the availability of an intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic constants from steady-state data. By employment of a minimal mechanism for bovine plasma amine oxidase involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations have been derived that allow calculation of k3 and K1 when DKeq congruent to 1 less than Dk. Unexpectedly, in the case of K1, we have shown that this parameter can be calculated from steady-state parameters without the requirement for an intrinsic isotope effect. This result should have general application to both ping-pong and sequential ternary-complex enzyme mechanisms. Of significance for future applications of steady-state isotope effects to the calculation of microscopic constants, values for K1 and k3 derived from steady-state parameters and single turnover measurements indicate excellent agreement. Compilation of parameters among six ring-substituted phenethylamines reveals alteration in delta G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C-H bond activation. A detailed discussion of the relevance of these findings to the interrelationship of binding energy and catalytic efficiency in enzyme reactions is presented.