Implications of the role of reactive cystein in arginine kinase: reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase reactivated by dithiothreitol.
The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.
Pan
Ji-Cheng
JC
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, PR China.
Cheng
Yuan
Y
Hui
En-Fu
EF
Zhou
Hai-Meng
HM
D001122:Arginine Kinase / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry*; D004793:Enzyme Reactivators / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D013379:Substrate Specificity
D004793:Enzyme Reactivators; D004228:Dithionitrobenzoic Acid; D001122:Arginine Kinase; D008274:Magnesium; D003545:Cysteine; D004229:Dithiothreitol