Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate:inorganic phosphate equilibration

Abstract

Two methods for quantitatively measuring PPi in the presence of 103-104 M excesses of Pi were developed and used to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. The rate of enzyme-catalyzed H2O-phosphate O2 exchange was measured. Both processes have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson, et al. (1979) that O2 exchange proceeds via EPPi formation. The results are consistent with a model in which 3 Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only 2 Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi-Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The equilibrium constant for PPi (enzyme-bound) .dblarw. 2Pi (enzyme-bound) is 4.8. Following PPi hydroylsis, the 1st released Pi contains an O from solvent water. The steps for PPi hydrolysis on the enzyme and for release of both product Pi''s are all partially rate-determining in overall enzyme-catalyzed PPi hydrolysis. PPi formation on the enzyme is rate determining for H2O-Pi O exchange. PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi-PPi exchange (Cohn, 1958; Janson, et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.