Siksnys V, Pleckaityte M
Institute of Biotechnology Fermentas, Vilnius, Lithuania.
Biochim Biophys Acta. 1992 Nov 20;1160(2):199-205. doi: 10.1016/0167-4838(92)90008-2.
Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.
进行了化学修饰研究，以阐明半胱氨酸残基在限制性内切酶Cfr9I的催化/结合过程中的作用。在pH 7.5条件下，将限制性内切酶Cfr9I与N-乙基马来酰亚胺（NEM）、碘乙酸盐、5,5'-二硫代双（2-硝基苯甲酸）一起孵育，导致催化活性完全丧失。然而，用碘乙酰胺和甲硫基磺酸盐修饰该酶后，未检测到酶失活。在底物存在的情况下，观察到该酶完全受到NEM失活的保护，这意味着半胱氨酸残基可能位于酶的活性位点或其附近。使用凝胶迁移率变动分析对天然和修饰的限制性内切酶Cfr9I进行直接底物结合研究表明，底物结合会阻碍NEM对该酶的修饰。在用5,5'-二硫代双（2-硝基苯甲酸）滴定该酶的过程中，一个半胱氨酸残基被修饰，同时催化活性丧失。NEM使Cfr9I失活的pH依赖性揭示了pKa值为8.9±0.2的残基被修饰。Cfr9I催化底物水解的反应速率对pH的依赖性揭示了两个必需残基分别具有pKa值6.3±0.15和8.7±0.15。所提供的证据表明，限制性内切酶Cfr9I含有一个反应性巯基残基，该残基对催化作用并非必需，但位于底物结合位点或其附近。
