Pinitglang S, Watts A B, Patel M, Reid J D, Noble M A, Gul S, Bokth A, Naeem A, Patel H, Thomas E W, Sreedharan S K, Verma C, Brocklehurst K
Laboratory of Structural and Mechanistic Enzymology, Department of Biochemistry, Queen Mary and Westfield College, University of London, London E1 4NS, U.K.
Biochemistry. 1997 Aug 19;36(33):9968-82. doi: 10.1021/bi9705974.
The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of these enzymes mirrors the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pKa value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic site thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pKa values for the formation and deprotonation of the (Cys)-S-/(His)-Im+H ion pair state. Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pKa values are compared with those observed in the pH dependence of kcat/Km for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pKa value for ion pair formation and the common pKa value close to 4 related to generation of catalytic activity is particularly marked for ficin (pKa 2.49 +/- 0.02) and caricain (pKa 2.88 +/- 0.02) but exists also for papain (pKa 3.32 +/- 0.01).
半胱氨酸蛋白酶超家族是机制研究中有价值的天然结构变体的来源。长期以来，人们一直认为这些酶的催化能力反映了普遍存在的催化位点咪唑硫醇离子对的形成，这是不言而喻的。然而，我们在此报告了动力学研究的确凿证据，并得到了静电势计算的支持，即至少对于其中一些酶来说，提供亲核和酸碱化学性质的离子对状态在酶无活性的低pH值下基本已完全形成。催化能力需要额外的质子解离，其共同的pKa值接近4，可能来自Glu50簇，以控制离子对的几何形状。催化位点硫醇基团与4,4'-二嘧啶基二硫化物反应的二级速率常数（k）的pH依赖性表明，它提供了（半胱氨酸）-S-/(组氨酸)-Im+H离子对状态形成和去质子化的pKa值。与2,2'-二吡啶基二硫化物反应的类似研究揭示了其他动力学上有影响的电离作用，并且将所有这些pKa值与在N-乙酰苯丙氨酰甘氨酸4-硝基苯胺催化水解的kcat/Km的pH依赖性中观察到的pKa值进行了比较。对于无花果蛋白酶（pKa 2.49±0.02）和木瓜凝乳蛋白酶（pKa 2.88±0.02），离子对形成的pKa值与接近4的与催化活性产生相关的共同pKa值之间的差异尤为明显，但木瓜蛋白酶（pKa 3.32±0.01）也存在这种差异。
