Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide.
Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R. F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H]NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate. Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.
Smyth
G E
GE
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D010449:Peptide Mapping; D013552:Swine; D014357:Trypsin
D009249:NADP; D007521:Isocitrate Dehydrogenase; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide