Reed P E, Katzenellenbogen J A
Department of Chemistry, University of Illinois, Urbana 61801.
J Biol Chem. 1991 Jan 5;266(1):13-21.
Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.
测试了含有原烯醇内酯和卤代烯醇内酯的脯氨酸 - 缬氨酸假二肽对丝氨酸蛋白酶人白细胞弹性蛋白酶（HLE）、猪胰弹性蛋白酶、α - 糜蛋白酶、胰蛋白酶、凝血酶和尿激酶的抑制活性。发现原烯醇内酯1a - c是HLE的底物，但却是较差的替代底物抑制剂。如通过为每种酶测定的结合常数（KI）、酰化速率（ka）、失活速率和分配比所示，溴代烯醇内酯反式异构体2a被发现是HLE和糜蛋白酶的非常有效的抑制剂。这种抑制剂对其靶酶HLE的特异性比单取代卤代烯醇内酯更好；我们将此归因于一种假二肽酰基酶，其结构与HLE的良好肽底物所采用的结构相似。溴代烯醇内酯2a对糜蛋白酶的失活是永久性的，但用亲核试剂肼处理后，HLE的失活部分可恢复，这表明内酯2a产生了两种失活的HLE。这些物种中较稳定的可能是酰基酶中亲电溴甲基酮对His - 57烷基化的结果，而较不稳定的、可被肼重新激活的物种可能是Asp - 102烷基化的结果，或者是最初形成的酰基酶中溴甲基酮基团水解形成新的、更稳定的酰基酶的结果。
