Baum E Z, Siegel M M, Bebernitz G A, Hulmes J D, Sridharan L, Sun L, Tabei K, Johnston S H, Wildey M J, Nygaard J, Jones T R, Gluzman Y
Molecular Biology Research Section, Wyeth-Ayerst Research, Lederle Laboratories, Pearl River, New York 10965, USA.
Biochemistry. 1996 May 7;35(18):5838-46. doi: 10.1021/bi952996+.
A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.
一种对称取代的二硫化合物CL13933被鉴定为人类巨细胞病毒UL80蛋白酶的有效抑制剂。根据抑制剂浓度观察到两种类型的被抑制蛋白酶。在高浓度下，CL13933与蛋白酶的半胱氨酸残基形成共价加合物。在较低浓度下，该化合物诱导Cys84与Cys87之间以及Cys138与Cys161之间形成特定的分子内二硫键。相比之下，Cys202未形成二硫键。蛋白酶还原后抑制作用逆转。UL80蛋白酶的五个半胱氨酸中的每一个都分别突变为丙氨酸。每个突变蛋白酶都保留了酶活性，但突变体C138A和C161A对CL13933的抑制具有抗性，这表明Cys138与Cys161之间形成二硫键是抑制作用的原因。这种二硫键显然不是由空气氧化诱导的。通过质谱检查野生型和五个突变蛋白酶的CL13933负载模式，发现Cys87、Cys138和Cys161残基与CL13933反应，并且每个残基的二硫键配对伙伴（分别为Cys84、Cys161和Cys138）能够通过硫醇-二硫键交换取代该化合物。讨论了这些反应性硫醇在蛋白酶中的可能意义。
