A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step.
The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013844:Thiazoles / Q000737:chemistry*; D013871:Thiones / Q000737:chemistry*
D002614:Chelating Agents; D004791:Enzyme Inhibitors; C419923:N-hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione; D013844:Thiazoles; D013871:Thiones; D003300:Copper; D004299:Dopamine beta-Hydroxylase