A. Betz et al., Inhibition of factor Xa by a peptidyl-alpha-ketothiazole involves two steps. Evidence for a stabilizing conformational change, BIOCHEM, 38(44), 1999, pp. 14582-14591
Recently, peptidylketothiazoles have been shown to be potent inhibitors of
proteases, but the details of the interaction have not yet been studied. In
the work presented here, the interaction of factor Xa, a coagulation prote
ase, with the transition state inhibitor BnSO2-D-Arg-Gly-Arg-ketothiazole (
C921-78) is characterized. C921-78 is a tight and selective inhibitor of th
e Coagulation protease factor Xa (K-d = 14 pM). The hydrolytic activity of
factor Xa was inhibited by C921-78 in a time-dependent manner. The rate-lim
iting step of the bimolecular combination of inhibitor and enzyme was compe
titive with the substrate. Conversely, the inhibitor could be displaced fro
m the active sire of the enzyme after exposure of the preformed complex to
an excess of substrate or to the active site inhibitor dansyl-Glu-Gly-Arg-c
hloromethyl ketone (DEGR-CMK) in a slow reaction. The formation of the C921
-78-factor Xa complex resulted in a 60% increase in the magnitude of the fl
uorescence emission spectrum. Rapid mixing of the enzyme and inhibitor prod
uces a monophasic fluorescence increase, compatible with spectral transitio
n in a single step. The rate constant for this reaction increased hyperboli
cally with the concentration of C921-78, but the amplitude remained constan
t. These results are consistent with the initial formation of an enzyme-inh
ibitor complex (EI),: followed by a unimolecular conversion of EI to EI* li
nked to a spectral transition. The rate constants of the isomerization prov
ide an estimate of 300000-fold stabilization. Thus, the inhibition of facto
r Xa by C921-78 follows a mechanism similar to that described classically f
or slow tight binding inhibitors. However, the two steps of the reaction ca
nnot be kinetically separated by the rapid equilibrium assumption, and ther
efore, the formation of EI is partially rate-limiting, too. The driving ene
rgy for the unusually fast isomerization step may result from the highly fa
vorable interactions of the inhibitor in the primary binding site.