Allen_1989_Biochemistry_28_135

The kinetics of substrate hydrolysis by pig liver esterase show activation by various substrates as well as activation by organic solvents (both Vmax and Km increase) [Barker, D.L., & Jencks, W.P. (1969) Biochemistry 8, 3890]. The trifluoromethyl ketones 1,1,1-trifluoro-4-phenylbutan-2-one (TPB) and 1,1,1-trifluoro-4-(p-hydroxyphenyl)butan-2-one (OH-TPB) are slow, tight binding inhibitors of pig liver esterase with Ki values of 6.8 X 10(-9) M and 6.0 X 10(-9) M, respectively. Acetonitrile, TPB, and OH-TPB as well as the substrates pNPA and ethyl lactate caused a 15-130-fold increase in the rate of association (kon), and dissociation (koff), of the enzyme--TPB complex. The value of Ki (koff/kon) did not change. The effect cannot be attributed to half-sites reactivity since an increase in koff of OH-TPB is also observed with enzyme monomers. The results are consistent with a model proposed for the catalytic reaction (Barker & Jencks, 1969) which invokes two binding sites on each esterase subunit, a catalytic site and an effector site. Occupation of the effector site can increase koff and kon for the inhibitors TPB and OH-TPB. Not all compounds which bind at the effector site increase koff. Butanol binds at the effector site but does not effect koff of TPB. The results also indicate that an aromatic or a hydrophobic structure and a carbonyl group are required for optimal interaction with the effector site.