Structural studies on yeast 3-phosphoglycerate kinase. Identification by immuno-affinity chromatography of one glutamyl residue essential for yeast 3-phosphoglycerate kinase activity. Its location in the primary structure.
3-Phosphoglycerate kinase is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosine ethyl ester. The coupling of 1 mol nitrotyrosine/mol enzyme is sufficient to inactivate the protein completely. A weak protection against inactivation is observed with each substrate added separately. In contrast, the complex ATP--3-phosphoglycerate--enzyme or ATP--Mg--3-phosphoglycerate--enzyme affords a considerable protection. The critical residue is identified as a glutamyl residue after isolation by immuno-affinity chromatography of nitrotyrosyl peptide resulting from exhaustive proteolytic digestion of the modified protein. In addition, the determination of the primary sequence of the C-terminal part of the protein leads to the location of the glutamyl residue at position eight from the C-terminus. We conclude that this glutamyl residue is situated in the domain which does not bind the nucleotide substrates [Bryant, T.N., Watson, H.C. and Wendell, P.L. (1974) Nature (Lond.) 247, 14--17]. Its role in the catalysis process is discussed.
D000595:Amino Acid Sequence; D002234:Carbodiimides / Q000494:pharmacology; D043424:Carboxypeptidase B; D002268:Carboxypeptidases; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D005971:Glutamates / Q000032:analysis / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D002234:Carbodiimides; D005971:Glutamates; D010446:Peptide Fragments; D014443:Tyrosine; C024960:nitrotyrosine ethyl ester; D010735:Phosphoglycerate Kinase; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin; D003488:Cyanogen Bromide