Reaction of the sulphydryl groups of lobster-muscle glyceraldehyde-3-phosphate dehydrogenase with organic mercurials

Abstract

1. 1.|Crystalline lobster-muscle glyceraldehyde-3-posphate dehydrogenase ( d-glyceraldehyde-3-phosphate: NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) possesses 7.3 ± 0.3 cysteine residues per mole of enzyme (140 000 g), i.e. approx. 2 cysteine residues per polypeptide chain, which are extremely reactive toward organic mercurials; 8 of the 20 cysteine residues of the enzyme are totally unreactive toward organic mercurials. 2. 2.|Enzyme carboxymethylated at each of the 4 ‘active site’ cysteine residues possesses 7.4 ± 0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate and cannot be distinguished from native enzyme on the basis of these titrations. 3. 3.|Enzyme treated with iodosobenzoic acid and containing an intramolecular disulphide bridge at the ‘active center’ possesses 4.0 ± 0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate, i.e. approx. 4 less than native enzyme. 4. 4.|Under normal conditions, lobster-muscle glyceraldehyde-3-phosphate dehydrogenase is 90–95% inactivated concomitant with the formation of 8 moles of mercaptide per mole of enzyme (140 000 g); inhibition takes place in a linear manner with respect to mercurial concentration. 5. 5.|In the presence of a large molar excess of NAD +, the formation of 4 moles of mercaptide per mole of enzyme (140 000 g) has no inhibitory effect and must be attributed to reaction of a ‘nonessential’ sulphydryl group.