Studies with human erythrocyte pyruvate kinase (PK): effects of modification of sulfhydryl groups

Cysteinyl residues of red cell pyruvate kinase (PK; ATP: pyruvate phosphotransferase, EC 2.7.1.40) were modified with methylmethanethiosulfonate (MMTS), p-nitrophenoxycarbonyl methyl disulfide (NPCMD), and sodium tetrathionate (NaTT). At pH greater than 7 . 0, K0.5 s phosphoenol-pyruvate (PEP) was markedly increased. Fructose-1,6-diphosphate (FDP) increased affinity for PEP, but K0.5 s (PEP) remained elevated and hyperbolic kinetics were not achieved. Inhibition by negative effectors ATP and alanine was not reversed by PEP and FDP concentrations far greater than those abolishing inhibition of unmodified enzyme. At pH less than 7 . 0, PEP affinity was reduced, and FDP markedly increased Vmax and diminished K0.5 s (PEP). MMTS greatly impaired the thermostability of PK. Acid pH alone and the simultaneous presence of Mg++, K+ and PEP prior to MMTS treatment protected against the effects on PEP kinetics, but did not alter the induction of thermolability. No MMTS effect on the FDP binding site, on ADP kinetics or on the relative effectiveness of GDP, UDP or CDP cofactors was demonstrated. The MMTS-induced alterations closely resembled those observed with certain PK mutants associated with haemolytic anaemia.