Bowles M R, King G J, Berndt M C, Zerner B
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Biochim Biophys Acta. 1996 Dec 5;1298(2):167-79. doi: 10.1016/s0167-4838(96)00116-1.
The reactions of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone (TDPP) with chicken liver carboxylesterase have shown that this ketone hydrate is not only a potent inhibitor of the enzyme, but also a substrate for a number of enzyme-catalyzed reactions. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is initially dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent ketone then produces a hemiketal adduct. However, the slow reactivation (by dialysis) of TDPP-inhibited enzyme indicates that the interaction with this inhibitor is more complex. At equilibrium, a dissociation constant of 2.4 pM was obtained for this interaction. 19F-NMR studies of the enzyme-TDPP complex show that after pre-equilibration, the major adduct is not the hemiketal adduct. It is proposed that this final adduct is a cross-linked adduct formed between TDPP, the active-site serine and the active-site histidine. 19F-NMR studies reveal that chicken liver carboxylesterase catalyses the cleavage of TDPP to yield either fluoride ion or trifluoroacetate, and also the benzilic acid rearrangement of TDPP to alpha-trifluoromethylmandelate. These products have also been identified in model studies of the reaction between TDPP and imidazole.
3,3,3-三氟-2,2-二羟基-1-苯基-1-丙酮（TDPP）与鸡肝羧酸酯酶的反应表明，这种酮水合物不仅是该酶的强效抑制剂，也是多种酶催化反应的底物。抑制动力学与一种机制相符，即结合的水合物最初在酶催化的限速步骤中脱水。然后，活性位点丝氨酸对母体酮的亲核攻击产生半缩酮加合物。然而，TDPP抑制的酶通过透析缓慢重新激活表明与该抑制剂的相互作用更为复杂。在平衡时，这种相互作用的解离常数为2.4 pM。对酶-TDPP复合物的19F-NMR研究表明，预平衡后，主要加合物不是半缩酮加合物。有人提出，这种最终加合物是TDPP、活性位点丝氨酸和活性位点组氨酸之间形成的交联加合物。19F-NMR研究表明，鸡肝羧酸酯酶催化TDPP裂解生成氟离子或三氟乙酸盐，还催化TDPP重排为α-三氟甲基扁桃酸。这些产物也在TDPP与咪唑反应的模型研究中得到了鉴定。
