Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by oxidation of a specific sulfhydryl group with potassium permanganate.
Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003201:Computers; D003544:Cysteic Acid / Q000378:metabolism; D003553:Cystine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D011196:Potassium Permanganate / Q000494:pharmacology*; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology; D012486:Salmonella typhimurium / Q000201:enzymology; D013438:Sulfhydryl Compounds; D013793:Thallium / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D014461:Ultracentrifugation
D000596:Amino Acids; D009579:Nitrobenzoates; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D012267:Ribosemonophosphates; D013438:Sulfhydryl Compounds; D011196:Potassium Permanganate; D014443:Tyrosine; D003553:Cystine; D006639:Histidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003544:Cysteic Acid; D013793:Thallium; D008715:Methionine; D010770:Phosphotransferases; D012268:Ribose-Phosphate Pyrophosphokinase; D008274:Magnesium