Yeast inorganic pyrophosphatase substrate recognition

Monodentate Co(NH₃)₅PP_i was determined not to be a substrate for yeast inorganic pyrophosphatase while P¹,P²-bidentate Co(NH₃)₄PP_i 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)₂PP_i, Cr(NH₃)₄PP_i, Cr(H₂O)(NH₃)₃PP_i, Cr(H₂O)₂(NH₃)₂PP_i, and Cr(H₂O)₄PP_i 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 K_m for Mg²⁺, the binding affinity of the complexes decreased with an increasing $\text{NH}{}_3\text{H}{}_2\text{O}$ ligand ratio as did the turnover number of the corresponding central complexes. The Co(en)₂PP_i complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH₃)₄PP_i complex. The substrate activities of β,γ-bidentate Co(NH₃)₄PPP_i and α,β,γ-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.