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Studies with human erythrocyte pyruvate kinase (PK): effects of modification of sulfhydryl groups
Author(s) -
Valentine William N.,
Paglia Donald E.
Publication year - 1983
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1983.tb02039.x
Subject(s) - pyruvate kinase , chemistry , kinetics , biochemistry , glycolysis , pkm2 , enzyme , physics , quantum mechanics
S ummary . 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>iddot;0, K 0·5 s phosphoenol‐pyruvate (PEP) was markedly increased. Fructose‐1, 6‐diphosphate (FDP) increased affinity for PEP, but K 0·5 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 < 7·0, PEP affinity was reduced, and FDP markedly increased V max and diminished K 0·5 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.