Miwa N, Sawada T, Suzuki A
Eur J Biochem. 1984 May 2;140(3):539-45. doi: 10.1111/j.1432-1033.1984.tb08135.x.
A single SH group in the B chain (33 kDa), generated by the specific reduction of the single interchain SS bond of human urinary urokinase, was alkylated (UK X B) with iodoacetamide to prevent a spontaneous SH-SS interchange. An SS bond in UK X B was exclusively alkylated with iodoacetamide (R X CAM-UK X B) after reduction with dithiothreitol in 0.3 M guanidine X HCl in the presence of the competitive inhibitor N alpha-benzoyl-L- argininamide with concomitant loss of 65-68% of the esterolytic activity towards N-acetyl-glycyl-L-lysine methyl ester. This specific SS bond was located at Cys194 - Cys222 whose SS loop contained the active-site Ser198 , as determined by amino acid analyses and identification of the N and C termini of the tryptic digest. Transformation of UK X B into R X CAM-UK X B induced no shift of the optimal pH in the bell-shaped pH/activity profile; pH values for 50% activity were similar (pH 9.7) for 10-min alkalization of the enzyme but different between UK X B (pH 9.4) and R X CAM-UK X B (pH 8.8) for 18-h alkalization. An unaltered Km value and a decline by 64% in kcat in the esterolytic activity indicate that the pretransition Michaelis complex is formed without degeneration of the primary substrate-binding site, but the catalytic pathway thereafter has deteriorated. In affinity labeling with dansyl chloride or N alpha-tosyl-L-lysine chloromethylketone, which interrupted the catalysis at the latest at a stage involving the abortive acyl intermediate, the second-order rate constant for UK X B was lowered to 28% or 35% for R X CAM-UK X B, respectively, but the labeling yields were similar. The results indicate that indispensable structural elements, such as the catalytic triad and oxyanion hole, are maintained but a local conformation, which is necessary for efficient transition to the acyl intermediate and/or for resistance against alkaline inactivation, is destabilized with Cys194 - Cys222 scission.
通过特异性还原人尿激酶的单个链间二硫键，在B链（33 kDa）中产生了一个巯基，用碘乙酰胺将其烷基化（UK X B），以防止自发的巯基-二硫键互换。在用二硫苏糖醇在0.3 M盐酸胍中还原后，在竞争性抑制剂Nα-苯甲酰-L-精氨酰胺存在下，UK X B中的一个二硫键被碘乙酰胺专门烷基化（R X CAM-UK X B），同时对N-乙酰甘氨酰-L-赖氨酸甲酯的酯解活性损失65 - 68%。通过氨基酸分析以及胰蛋白酶消化产物N端和C端的鉴定确定，这个特定的二硫键位于Cys194 - Cys222，其硫键环包含活性位点Ser198。UK X B转化为R X CAM-UK X B在钟形pH/活性曲线中未引起最佳pH的偏移；对于酶的10分钟碱化，50%活性的pH值相似（pH 9.7），但对于18小时碱化，UK X B（pH 9.4）和R X CAM-UK X B（pH 8.8）之间不同。酯解活性中未改变的Km值和kcat下降64%表明，预过渡米氏复合物的形成没有使主要底物结合位点退化，但此后的催化途径已经恶化。在用丹磺酰氯或Nα-甲苯磺酰-L-赖氨酸氯甲基酮进行亲和标记时，它们最晚在涉及无效酰基中间体的阶段中断催化，UK X B的二级速率常数分别降至R X CAM-UK X B的28%或35%，但标记产率相似。结果表明，诸如催化三联体和氧阴离子洞等不可或缺的结构元件得以保留，但对于有效转化为酰基中间体和/或抵抗碱性失活所必需的局部构象，随着Cys194 - Cys222的断裂而变得不稳定。
