Reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified creatine kinase reactivated by dithiothreitol.
The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified creatine by dithiothreitol has been studied using the kinetic theory of the substrate reaction during modification of enzyme activity as previously described by C.L. Tsou (Adv. Enzymol. Rel. Areas Mol. Biol. 61 (1988) 381-436). The results show that the modified creatine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analogue markedly slows the apparent reactivation rate constant, while creatine shows no effect. The substrates creatine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO-3 cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of creatine kinase are discussed. It is suggested that the cysteine may be located in the hinge area of the two domains of creatine kinase. The reactive cysteine of creatine kinase may play an important role not in the binding to the transition-state analogue but in the conformational changes caused by the transition-state analogue.
Yang
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Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Zhou
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003401:Creatine / Q000378:metabolism; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol / Q000494:pharmacology*; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000494:pharmacology*; D007700:Kinetics; D009566:Nitrates / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents
D004793:Enzyme Reactivators; D009566:Nitrates; D013439:Sulfhydryl Reagents; D000244:Adenosine Diphosphate; C031618:sodium nitrate; D004228:Dithionitrobenzoic Acid; D003402:Creatine Kinase; D003401:Creatine; D004229:Dithiothreitol