A Slow-Tight Binding Inhibitor of Dopamine -Monooxygenase: A Transition State Analogue for the Product Release Step

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• 作者：Sanjeewa P. Dharmasena ; D. Shyamali Wimalasena ; and Kandatege Wimalasena
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 15, 2002
• 年：2002
• 卷：41
• 期：41
• 页码：12414 - 12420
• 全文大小：107K
• 年卷期：v.41,no.41(October 15, 2002)
• ISSN：1520-4995

文摘

The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT)is a potent tight-binding inhibitor for dopamine -monooxygenase (DM) with a dissociation constant of0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometryof the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor withfixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. Theobserved kinetic behavior is consistent with the apparent direct formation of the tightly bound E·I* complex.The k_on and k_off for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitorconcentrations were found to be (1.85 ± 0.07) × 10⁶ M^-1 s^-1 and (1.9 ± 0.6) × 10^-3 s^-1, respectively.The dissociation constant calculated from these rates was similar to that determined under steady-stateconditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well aspre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitivewith respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized formof the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with theproposal that the E·3H4PTT complex may mimic the transition state for the product (protonated) releasestep of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the propertiesof the E·P complex of the DM reaction and also as an active site titrant for the oxidized enzyme.