Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase activity by dithiothreitol or 2-mercaptoethanol.
Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating crab-shell and threatens the survival of the animal. The present paper studies the effects of thiohydroxyal compounds on the activity of green crab alkaline phosphatase. The results show that thiohydroxyal compounds can lead to reversible inhibition. The equilibrium constants have been determined for dithiothreitol (DTT) and mercaptoethanol (ME) binding with the enzyme and/or the enzyme-substrate complexes. The results show that both DTT and ME are non-competitive inhibitors. The kinetics of enzyme inactivation by ME at low concentrations has been studied using the kinetic method of the substrate reaction. The results suggest that at pH 10.0, the action of ME on green crab ALP is first quick equilibrium binding and then slow inactivation. The microscopic rate constants were determined for inactivation and reactivation. The rate constant of the forward inactivation (k(+0)) is much larger than that of the reverse reactivation (k(-0)). Therefore, when the ME concentration is sufficiently large, the enzyme is completely inactivated.
Zhang
R Q
RQ
Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing, People's Republic of China.
Chen
Q X
QX
Zheng
W Z
WZ
Lin
J Y
JY
Zhuang
Z L
ZL
Zhou
H M
HM
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D003386:Brachyura / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology*; D008667:Metalloproteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism
D004791:Enzyme Inhibitors; D008667:Metalloproteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D008623:Mercaptoethanol; D000469:Alkaline Phosphatase; D004229:Dithiothreitol