The kinetic study of arginine kinase from the sea cucumber Stichopus japonicus with 5,5'-dithiobis-(2-nitrobenzoic acid).
The Stichopus japonicus arginine kinase (AK) is a significant dimeric enzyme. Its modification and inactivation course with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and the reactivation course of DTNB-modified AK by dithiothreitol were investigated on the basis of the kinetic theory of the substrate reaction during the modification of enzyme activity. The results show that the modification is a biphasic course while the inactivation is monophasic, with one essential reactive cysteine per subunit. The Cys274 (numbering from the Stichopus sequence) is exposed to DTNB and is near the ATP binding site. The modified AK can be 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. The analog components, 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 its role in the catalysis of AK are discussed. The results suggest that the cysteine may be located in the hinge area of the two domains of AK. The reactive cysteine of AK, which was proposed to be Cys274, may play an important role not in the binding of the transition-state analog but in the conformational changes caused by the transition-state analog.
Feng
Zhao
Z
Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Qin
Guo
G
Xicheng
Wang
W
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012616:Sea Cucumbers / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D013438:Sulfhydryl Compounds; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D001122:Arginine Kinase; D008274:Magnesium; D003545:Cysteine; D004229:Dithiothreitol