title: The role of cysteine residues in the catalytic activity of glycerol-3-phosphate dehydrogenase.
abstract: Glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxido-reductase, EC 1.1.1.8) has been shown to be sensitive to inhibition by iodoacetate. The reaction of the enzyme with iodoacetate, which appears to be a simple bimolecular process, is accompanied by a corresponding loss of enzyme activity. In addition to changes in activity, the alkylation reaction was monitored by the incorporation of radioactivity from iodo[2-14C]acetate, by changes in amino acid composition, and by changes in the content of free sulfhydryl groups. It is concluded that there are two cysteine residues in the native dimeric enzyme which are essential for enzymic activity. The rate of inactivation was relatively insensitive to the presence of various compounds with the exception of NADH which markedly inhibited the reaction. Kinetic and binding studies showed that the binding of NADH prevents alkylation and, conversely, alkylation prevents NADH binding. From the pH dependence of the alkylation reaction, the pKa of the essential sulfhydryl groups was calculated to be 8.5 and it is suggested that the binding of coenzyme is independent of the state of ionization of these groups.
mesh_terms: D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003545:Cysteine; D005993:Glycerolphosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D009243:NAD; D005993:Glycerolphosphate Dehydrogenase; D003545:Cysteine