McGrath W J, Abola A P, Toledo D L, Brown M T, Mangel W F
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Virology. 1996 Mar 1;217(1):131-8. doi: 10.1006/viro.1996.0100.
Virus-coded proteinases are attractive targets for antiviral therapy; however, lack of quick, sensitive, quantitative, and selective assays for enzyme activity makes it difficult to characterize these proteinases and to screen large numbers of potential inhibitors. Here we describe new substrates for the adenovirus proteinase, fluorogenic Rhodamine-based substrates containing tetrapeptides corresponding to sequences cleaved in adenovirus precursor proteins. Proteinase activity in as few as 10(9) disrupted virions could be quantitatively detected in a 30-min assay. With the substrate (Leu-Arg-Gly-Gly-NH)2-Rhodamine, the Km was 1.4 microM and the Vmax was 3.24 pmol substrate hydrolyzed/sec/pmol virus. Enzyme activity was stimulated by dithiothreitol and inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. In a thiol protection experiment, the virion enzyme was shown to have a cysteine residue with an unusually low pKa, a pKa similar to that of the active-site nucleophile of the cysteine proteinase papain. The curve of Vmax as a function of pH is unlike the curve from papain and implied that there are at least three ionizable groups whose protonation state can affect catalysis - one with a pKa of 6.2, another with a pKa of 7.2, and a third with a pKa of 8.3.
病毒编码的蛋白酶是抗病毒治疗的诱人靶点；然而，缺乏快速、灵敏、定量和选择性的酶活性检测方法使得难以对这些蛋白酶进行表征以及筛选大量潜在抑制剂。在此，我们描述了腺病毒蛋白酶的新底物，即基于罗丹明的荧光底物，其含有与腺病毒前体蛋白中被切割序列相对应的四肽。在30分钟的检测中，低至10⁹个裂解病毒颗粒中的蛋白酶活性都能够被定量检测。对于底物（Leu-Arg-Gly-Gly-NH）₂-罗丹明，Km为1.4微摩尔，Vmax为3.24皮摩尔底物水解/秒/皮摩尔病毒。酶活性受到二硫苏糖醇的刺激，并被几种丝氨酸特异性以及半胱氨酸特异性蛋白酶抑制剂所抑制。在一个硫醇保护实验中，病毒颗粒酶显示有一个pKa异常低的半胱氨酸残基，其pKa与半胱氨酸蛋白酶木瓜蛋白酶的活性位点亲核试剂的pKa相似。Vmax作为pH函数的曲线不同于木瓜蛋白酶的曲线，这意味着至少有三个可电离基团，其质子化状态能够影响催化作用——一个pKa为6.2，另一个pKa为7.2，第三个pKa为8.3。
