Allen K N, Abeles R H
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Biochemistry. 1989 Jan 10;28(1):135-40. doi: 10.1021/bi00427a020.
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.
猪肝酯酶催化底物水解的动力学表现出多种底物的激活作用以及有机溶剂的激活作用（Vmax和Km均增加）[Barker, D.L., & Jencks, W.P. (1969) Biochemistry 8, 3890]。三氟甲基酮1,1,1-三氟-4-苯基丁-2-酮（TPB）和1,1,1-三氟-4-（对羟基苯基）丁-2-酮（OH-TPB）是猪肝酯酶的缓慢、紧密结合抑制剂，其Ki值分别为6.8×10⁻⁹ M和6.0×10⁻⁹ M。乙腈、TPB、OH-TPB以及底物对硝基苯乙酸酯（pNPA）和乳酸乙酯使酶 - TPB复合物的缔合速率（kon）和解离速率（koff）增加了15 - 130倍。Ki（koff/kon）的值没有变化。这种效应不能归因于半位点反应性，因为在酶单体中也观察到OH-TPB的koff增加。结果与催化反应提出的模型（Barker & Jencks, 1969）一致，该模型在每个酯酶亚基上调用两个结合位点，一个催化位点和一个效应位点。效应位点的占据可以增加抑制剂TPB和OH-TPB的koff和kon。并非所有在效应位点结合的化合物都会增加koff。丁醇在效应位点结合，但不影响TPB的koff。结果还表明，与效应位点的最佳相互作用需要芳香或疏水结构以及羰基。
