The steady-state kinetic data show that 3-hydroxy-4-
phenylthiazole-2(3
H)-thione (3H4PTT)is a
potent tight-binding inhibitor for do
pamine
-monooxygenase (D
M) with a dissociation constant of0.9 nM. Ackermann-Potter
plots of the enzyme de
pendence 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 a
pparent direct formation of the tightly bound E·I* com
plex.The
kon and
koff for 3H4PTT which were determined under
pre-steady-state conditions at variable inhibitorconcentrations were found to be (1.85 ± 0.07) × 10
6 M
-1 s
-1 and (1.9 ± 0.6) × 10
-3 s
-1, res
pectively.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 as
pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is com
petitivewith res
pect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized formof the enzyme. The kinetic behavior and the structural
pro
perties of 3H4PTT are consistent with the
pro
posal that the E·3H4PTT com
plex may mimic the transition state for the
product (
protonated) releaseste
p of the enzyme. Therefore, 3H4PTT could be used as a convenient
probe to examine the
pro
pertiesof the E·P com
plex of the D
M reaction and also as an active site titrant for the oxidized enzyme.