Yeast inorganic pyrophosphatase substrate recognition.

Monodentate Co(NH3)5PPi was determined not to be a substrate for yeast inorganic pyrophosphatase while P1,P2-bidentate Co(NH3)4PPi was turned over by the enzyme at a rate of 7.5 min-1. A kinetic analysis of the substrate activities of the P1,P2-bidentate complexes, Co(en)2PPi, Cr(NH3)4PPi, Cr(H2O)(NH3)3PPi, and Cr(H2O)2(NH3)2PPi, and Cr(H2O)4PPi was carried out in order to access the potential role of the metal-water ligands in productive binding. While substitution of the H2O ligands with NH3 ligands had a minimal affect on the Km for Mg2+, the binding affinity of the complexes decreased with an increasing NH3/H2O ligand ratio as did the turnover number of the corresponding central complexes. The Co(en)2PPi complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH3)4PPi complex. The substrate activities of beta, gamma-bidentate Co(NH3)4PPPi and alpha, beta, gamma-tridentate Co(NH3)3PPP with pyrophosphatase were also tested. While both complexes were shown to bind tightly to the Mg2+-activated enzyme neither was hydrolyzed. On the other hand, in the presence of the Zn2+-activated enzyme the tridentate complex was turned over at a rate of 0.17 min-1 while the bidentate complex remained inert to hydrolysis.