fumarates and dimethyl-4-phenylenediamine

The interactions of reductants with dopamine beta-monooxygenase (DbetaM) were examined using two novel classes of reductants. The steady-state kinetics of the previously characterized DbetaM reductant, N,N-dimethyl-1,4-p-phenylenediamine (DMPD), were parallel to the ascorbic acid-supported reaction with respect to pH dependence and fumarate activation. DMPD also displayed pH and fumarate-dependent apparent negative cooperativity demonstrating that the previously reported cooperative behavior of DbetaM toward the reductant is not unique to ascorbic acid. The 6-OH phenyl and alkylphenyl-substituted ascorbic acid derivatives were more efficient reductants for the enzyme than ascorbic acid. Kinetic studies suggested that these derivatives behave as pseudo bisubstrates with respect to ascorbic acid and the amine substrate. The lack of apparent cooperative behavior with these derivatives suggests that this behavior of DbetaM is not common for all the reductants. Based on these findings and additional kinetic evidence, the proposal that the apparent negative cooperativity in the interaction of ascorbic acid with DbetaM was due to the presence of a distinct allosteric regulatory site has been ruled out. In contrast to previous models, where fumarate was proposed to interact with a distinct anion binding site, the effect of fumarate on the steady-state kinetics of these novel reductants suggests that fumarate and the reductant may interact with the same site of the enzyme. In accordance with these observations and mathematical analysis of the experimental data, a unified model for the apparent negative cooperativity and fumarate activation of DbetaM in which both fumarate and the reductant interact with the same site of all forms of the enzyme with varying affinities under steady-state turnover conditions has been proposed.

Topics: Anticarcinogenic Agents; Chromatography, High Pressure Liquid; Dopamine beta-Hydroxylase; Enzyme Activation; Fumarates; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Phenylenediamines