Stein R L, Trainor D A
Biochemistry. 1986 Sep 23;25(19):5414-9. doi: 10.1021/bi00367a011.
The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了氯甲基酮MeOSuc-Ala-Ala-Pro-Val-CH2Cl对人白细胞弹性蛋白酶（HLE）的失活机制。失活的一级速率常数对氯甲基酮浓度的依赖性呈双曲线，表明在酶与抑制剂发生共价相互作用之前形成了可逆的“米氏复合物”。然而，观察到的Ki值为10 microM，至少比HLE与结构相关底物或可逆抑制剂相互作用形成的复合物的解离常数低10倍，这表明Ki是一个复杂的动力学常数，反映了米氏复合物和第二种复合物的形成与积累。有人提出，这种第二种复合物是由活性位点丝氨酸攻击抑制剂的羰基碳形成的半缩酮。这种中间体的积累可能是丝氨酸蛋白酶与特定肽衍生的氯甲基酮反应的一个普遍特征，并解释了这些反应中观察到的极低的Ki值。失活步骤（ki）的溶剂氘同位素效应（SIE）为1.58±0.07，与活性位点组氨酸对抑制剂亚甲基碳的限速、一般催化攻击并伴随氯离子阴离子取代一致。一般催化剂被认为是活性位点天冬氨酸。相比之下，HLE失活的二级速率常数ki/Ki的SIE是相反的，等于0.64±0.05。（摘要截短于250字）
