EFFECT OF PH ON TRANSIENT REDUCTION OF PIG-PLASMA BENZYLAMINE OXIDASE BY BENZYLAMINE DERIVATIVES

The transient kinetics of reduction of 470-nm absorption band in [pig plasma] benzylamine oxidase [EC 1.4.3.4] by substrate at different pH values between 6 and 10 were studied by stopped-flow techniques. Substituent effects on kinetic parameters from the reduction process were examined using a series of ring-substituted benzylamine derivatives as the substrates. Enzymatic reduction by substrate occurs in 2 kinetically distinguishable steps, with the intermediate formation of an enzyme-substrate complex in which the substrate appears to be covalently bound through its amino group to the prosthetic group of the enzyme, possibly in the form of an amine-pyridoxal Schiff-base. The apparent stability of the enzyme-substrate complex shows no dependence on the electronic properties of the amine substrates, but is strongly pH-dependent, suggesting the substrate-binding involves the non-protonated amines, exclusively, and requires the presence of the acid form of an ionizing group in the enzyme with apparent pKa of 8.8. Reduction of the enzymatic 470-nm chromophore and release of the aldehyde product of the catalytic process are rate-limited by the same monomolecular reaction step involving the enzyme-substrate complex. Rate constants for the rate-limiting reaction exhibit no significant dependence on pH between 6 and 10, but correlate with Hammett .sigma.-values for the ring-substituted benzylamine derivatives tested, yielding a .rho.-value of +0.3.