Chemical modification of rat liver cytosolic NADP(+)-linked isocitrate dehydrogenase by N-ethylmaleimide. Evidence for essential sulphydryl groups.
Incubation of rat liver cytosolic isocitrate dehydrogenase with N-ethylmaleimide (NEM) resulted in the inactivation of the enzyme following pseudo-first order kinetics. Isocitrate affords considerable protection against inactivation whereas NADP+ enhances modification of the enzyme, suggesting localization of the modified group at the active site. Correlation of loss of activity with incorporation of [14C]NEM indicated that two sulphydryl residues/sub-unit are modified of which only one is shown to be involved in catalysis. pH dependence of the inactivation process implicates a reactive group of pKa 8.1 in catalysis. We conclude that a unique cysteine residue is essential for maximal catalytic activity of isocitrate dehydrogenase.
Fatania
H R
HR
Department of Biochemistry, Faculty of Medicine, Kuwait University.
al-Nassar
K E
KE
Thomas
N
N
D000818:Animals; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008968:Molecular Conformation; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism
D013438:Sulfhydryl Compounds; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D003545:Cysteine; D005033:Ethylmaleimide