Glutamic acid-149 is important for enzymatic activity of yeast inorganic pyrophosphatase.
Modification of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is known to lead to a loss of enzymatic activity, the rate of which is decreased in the presence of ligands binding to the active site [Cooperman, B. S., & Chiu, N. Y. (1973) Biochemistry 12, 1676-1682; Heitman, P., & Uhlig, H. J. (1974) Acta Biol. Med. Ger. 32, 565-594]. In this work we show that, when such inactivation is carried out in the presence of [14C]glycine ethyl ester (GEE), GEE is covalently incorporated into PPase, incorporation into the most highly labeled tryptic peptide is site-specific, as evidenced by the reduction of such incorporation in the presence of the active site ligands Zn2+ and Pi, the extent of formation of this specifically labeled peptide correlates with the fractional loss of PPase activity, and the specifically labeled peptide corresponds to residues 145-153 and the position of incorporation within this peptide is Glu-149. The significance of our findings for the location of the active site and for the catalytic mechanism of PPase is briefly considered in the light of the 3-A X-ray crystallographic structure of Arutyunyun and his colleagues [Arutyunyun, E. G., et al. (1981) Dokl. Akad. Nauk SSSR 258, 1481-1485; Kuranova, I. P., et al. (1983) Bioorg. Khim. 9, 1611-1919; Terzyan, S. S., et al. (1984) Bioorg. Khim. 10, 1469-1482].
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005971:Glutamates; D018698:Glutamic Acid; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000596:Amino Acids; D002234:Carbodiimides; D005971:Glutamates; D008024:Ligands; D010446:Peptide Fragments; D018698:Glutamic Acid; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D005022:Ethyldimethylaminopropyl Carbodiimide