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

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Abstract
Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as 2 half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by O2. Anaerobic stopped-flow kinetic measurements of the 1st 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.sbd.H bond cleavage step, k3, directly. Steady-state isotope effects were also measured for benzylamine and 6 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 .times. 102 to 3.8 .times. 104 M-1 s-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier, 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 involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations were derived that allow calculation of k3 and K1 and dKeq .simeq. 1 < Dk. Unexpectedly, in the case of K1, 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 significnace 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 6 ring-substituted phenethylamines reveales alteration in .DELTA.G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C.sbd.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.