The effects of anions, substrates, metal ions and sulfhydryl reagents on the proteolytic susceptibility of yeast phosphoglycerate kinase.
With the aim of confirming our previous spectrophotometric binding studies ((1978) Eur. J. Biochem. 85, 345-350 and (1980) Eur. J. Biochem. 104, 249-254) and of ascertaining the full physiological significance of ion binding, we investigated the effects of ions and thiol reagents on the proteolysis of yeast phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). The single non-essential thiol of the enzyme was modified with 5,5'-dithiobis(2-nitrobenzoic acid) or 2-chloromercuri-4-nitrophenol. Both modifications greatly increased the susceptibility of the kinase to inactivation by trypsin or yeast proteinase A, when compared with that of the native kinase. Electrophoresis in sodium dodecyl sulfate (SDS) revealed that limited proteolysis had occurred. The time courses for the proteolysis and loss of catalytic activity were followed and the active and inactive fragments identified. The molecular masses of the major proteolytic fragments differed with the two endopeptidases. Substrate and non-substrate anions in a concentration-dependent fashion, protected the native and mercurial-labelled kinase from inactivation by trypsin or yeast proteinase A. However, Zn2+, in a concentration-dependent fashion, increased the susceptibility of the native kinase to inactivation by each endopeptidase. The time courses for the inactivation and for the proteolysis allowed the active and inactive fragments to be identified. Zn2+ decreased the rate of inactivation of the mercurial-labelled kinase by proteinase A. The effects of these ions were detected at concentrations compatible with occupancy of an anion binding site and a low affinity Zn2+ binding site, both of which have been indicated from our previous binding studies.
D000838:Anions; D016282:Aspartic Acid Endopeptidases; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013386:Succinates / Q000494:pharmacology*; D019802:Succinic Acid; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D000838:Anions; D002951:Citrates; D013386:Succinates; D013439:Sulfhydryl Reagents; D019343:Citric Acid; D004228:Dithionitrobenzoic Acid; D019802:Succinic Acid; D010735:Phosphoglycerate Kinase; D010450:Endopeptidases; D014357:Trypsin; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; D015032:Zinc