title: Yeast inorganic pyrophosphatase. A model for active-site structure based on 113Cd2+ and 31P NMR studies.
abstract: Equilibrium dialysis and 113Cd2+ NMR studies in the presence of inorganic phosphate (Pi) provide clear evidence for the existence of four well-defined Cd2+ sites per yeast inorganic pyrophosphatase subunit. Parallel 31P NMR studies demonstrate the existence of two binding sites per subunit for Pi and provide strong confirmatory evidence for a small amount of enzyme-bound inorganic pyrophosphate in equilibrium with enzyme-bound Pi. Such inorganic pyrophosphate formation was demonstrated by chemical analysis earlier [Welsh, K.M., Armitage, I.M., & Cooperman, B.S. (1983) Biochemistry 22, 1046-1054]. In this same earlier paper, we provided evidence for inner-sphere contact between enzyme-bound Cd2+ and Pi bound in the higher affinity of the two Pi sites. We now present heteronuclear decoupling evidence that one to three different Cd2+ ions make such contact. The divalent metal ion cofactor conferring the highest activity on inorganic pyrophosphatase is Mg2+, and we present evidence from competition experiments that such inner-sphere contact is also likely for the Mg2+-enzyme. On the other hand, these experiments also show that some metal ion binding sites on the enzyme bind Mg2+ and not Cd2+, and some bind Cd2+ and not Mg2+. These results are considered along with others obtained recently in proposing an active-site structure for inorganic pyrophosphatase.
mesh_terms: D001665:Binding Sites; D002104:Cadmium / Q000032:analysis / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D010710:Phosphates; D002104:Cadmium; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase