Interaction of glyceraldehyde-3-phosphate dehydrogenase with SH-containing compounds: evidence for the binding of L-cysteine and for the dependence of the binding on the functional state of the enzyme.
Incorporation of L-[35S]cysteine into rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was detected following incubation of the enzyme in a mixture containing glyceraldehyde-3-phosphate, NAD+ and the labeled cysteine. Insignificant binding occurred in the absence of either the substrate or NAD+, suggesting that formation of an acylated enzyme form was a prerequisite for the binding. Stoichiometry of the binding depended on the number of functioning active centers; up to 4 moles of L-[35S]cysteine bound per mole tetramer with fresh enzyme preparations. The L-[35S]cysteine incorporation depended on pH and was maximal when a group having pKa of 8.5 is protonated. To clarify the relevance of this finding to the effect of SH-containing compounds previously shown to decrease the rate of 3-phosphoglyceroyl-enzyme hydrolysis [Kuzminskaya et al., FEBS Lett. 336 (1993) 208-210], the pH-dependence of the effect of glutathione on the hydrolysis rate was determined and found to be close to the pH-dependence of L-[35S]cysteine binding.
D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D004800:Enzymes, Immobilized; D013438:Sulfhydryl Compounds; D013462:Sulfur Radioisotopes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine