Prorok M, Albeck A, Foxman B M, Abeles R H
Brandeis University Graduate Department of Biochemistry, Waltham, Massachusetts 02254.
Biochemistry. 1994 Aug 16;33(32):9784-90. doi: 10.1021/bi00198a050.
We have examined the reaction of N-(benzyloxycarbonyl)-L-alanyl-L-glycyl-L-phenylalanyl chloromethyl ketone (ZAGFCMK) with chymotrypsin (Cht) and have found that, in addition to irreversible alkylation of the enzyme, some of the corresponding hydroxymethyl ketone is produced. For each molecule of hydroxy ketone formed, 3.6 molecules of chymotrypsin are inactivated. Chloroketone hydrolysis is also observed with chymotrypsin methylated at N-3 of the active site histidine (MeCht). The hydrolysis proceeds slowly (k = 0.14 min-1). Alkylation of the modified enzyme was not observed. An initial burst of free chloride is detected during the MeCht-catalyzed hydrolysis. The magnitude of the chloride burst is proportional to the enzyme concentration in an approximate 1:1 stoichiometry and indicates a relatively rapid chloride-releasing step which gives rise to an intermediate which is more slowly converted to hydroxy ketone. We have also investigated both the solution and MeCht-mediated hydrolysis of the S isomer of N-acetyl-L-alanyl-L-phenylalanyl chloroethyl ketone (S-AcAFCEK). We have concluded that the nonenzymatic hydrolysis proceeds with inversion of configuration at the stereocenter, while the enzymatic process occurs with retention of configuration. The two nucleophilic displacements attending the MeCht-mediated hydrolysis of S-AcAFCEK imply the formation of an intermediate, possibly of an epoxy ether, formed by internal displacement of the chloride by the oxyanion of the initially generated enzyme-chloroketone hemiketal adduct.
我们研究了N-（苄氧羰基）-L-丙氨酰-L-甘氨酰-L-苯丙氨酰氯甲基酮（ZAGFCMK）与胰凝乳蛋白酶（Cht）的反应，发现除了该酶发生不可逆烷基化外，还生成了一些相应的羟甲基酮。每形成一分子羟酮，就有3.6分子胰凝乳蛋白酶失活。在活性位点组氨酸的N-3位甲基化的胰凝乳蛋白酶（MeCht）也能观察到氯酮水解。水解过程进行得很慢（k = 0.14 min⁻¹）。未观察到修饰酶的烷基化。在MeCht催化的水解过程中检测到有游离氯离子的初始爆发。氯离子爆发的幅度与酶浓度成正比，化学计量比约为1:1，这表明有一个相对快速的氯离子释放步骤，产生了一个中间体，该中间体向羟酮的转化较为缓慢。我们还研究了N-乙酰-L-丙氨酰-L-苯丙氨酰氯乙基酮（S-AcAFCEK）的S异构体在溶液中和MeCht介导下的水解。我们得出结论，非酶水解在立体中心发生构型翻转，而酶促过程则构型保持不变。MeCht介导的S-AcAFCEK水解过程中的两个亲核取代反应意味着形成了一个中间体，可能是环氧醚，它是由最初生成的酶-氯酮半缩酮加合物的氧负离子对氯离子进行分子内取代而形成的。
