Lin G, Liao W C, Chiou S Y
Departnment of Chemistry and Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan.
Bioorg Med Chem. 2000 Nov;8(11):2601-7. doi: 10.1016/s0968-0896(00)00196-6.
4-Nitrophenyl-N-substituted carbamates (1-6) are the pseudo-substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the first step of the inhibition (Ki step) is the formation of the enzyme inhibitor tetrahedral adduct and the second step of the inhibition (kc) is the formation of the carbamyl enzyme. The formation of the enzyme inhibitor tetrahedral adduct is further divided into two steps, the formation of the enzyme-inhibitor complex with the dissociation constant, KS, at the first step and the formation of the enzyme-inhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for the formation of the enzyme-inhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitative structure-activity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 1-6 indicate that values of -logKs and logk2/k-2 are correlated with the Taft substituent constant, sigma*, and the rho* values from these correlations are -0.33 and 0.1, respectively. The negative rho* value for the -logKS-sigma*-correlation indicates that the first step of the two-step formation of the enzyme-inhibitor tetrahedral adduct (KS step) is the formation of the positive enzyme inhibitor complex. The positive rho* value for the logk2/k-2 -sigma*-correlation indicates that the enzyme inhibitor tetrahedral adduct is more negative than the enzyme inhibitor complex. Finally, the two-step mechanism for the formation of the enzyme inhibitor tetrahedral adduct is proposed according to these results. Thus, the partially positive charge is developed at nitrogen of carbamates 1-6 in the enzyme-inhibitor complex probably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 1-6 and the amide hydrogen of the oxyanion hole of the enzyme. The second step of the two-step formation of the enzyme-inhibitor tetrahedral adduct is the nucleophilic attack of the serine of the enzyme to the carbonyl group of carbamates 1-6 in the enzyme-inhibitor complex and develops the negative-charged oxygen in the adduct.
4-硝基苯基-N-取代氨基甲酸酯（1-6）是猪胰胆固醇酯酶的伪底物抑制剂。因此，抑制的第一步（Ki步骤）是酶抑制剂四面体加合物的形成，抑制的第二步（kc）是氨甲酰酶的形成。酶抑制剂四面体加合物的形成进一步分为两个步骤，第一步是形成具有解离常数KS的酶-抑制剂复合物，第二步是从该复合物形成酶-抑制剂四面体加合物。酶-抑制剂四面体加合物形成的两步机制通过预稳态动力学得到证实。氨基甲酸酯1-6对胆固醇酯酶预稳态抑制的定量构效关系结果表明，-logKs和logk2/k-2值与塔夫脱取代基常数sigma相关，这些相关性的rho值分别为-0.33和0.1。-logKS-sigma相关性的负rho值表明，酶-抑制剂四面体加合物形成的两步中的第一步（KS步骤）是正酶抑制剂复合物的形成。logk2/k-2 -sigma相关性的正rho值表明，酶抑制剂四面体加合物比酶抑制剂复合物更负。最后，根据这些结果提出了酶抑制剂四面体加合物形成的两步机制。因此，在酶-抑制剂复合物中，氨基甲酸酯1-6的氮上可能由于氨基甲酸酯1-6的氮孤对与酶的氧负离子孔的酰胺氢之间的氢键作用而产生部分正电荷。酶-抑制剂四面体加合物形成的两步中的第二步是酶的丝氨酸对酶-抑制剂复合物中氨基甲酸酯1-6的羰基进行亲核攻击，并在加合物中产生带负电荷的氧。
