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

Abstract

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two 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 major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) 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; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) 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.

Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements

“…Fluoride (Table II), in agreement with previous estimates (5,26,27). This remarkable feature of the active site was completely retained in D117E and D120E variants, partially retained in the D48E, Y93F, and D115E variants and almost completely lost in the D152E variant (Table II and Fig.…”

Dissociation of Hexameric Escherichia coli Inorganic Pyrophosphatase into Trimers on His-136 → Gln or His-140 → Gln Substitution and Its Effect on Enzyme Catalytic Properties

“…Variant PPases-Following the approach devised for S. cerevisiae PPase (Springs et al, 1981), we determined the rate and equilibrium constants of Scheme I for the H136Q-and H140Q-PPases by combining data from equilibrium formation of enzyme-bound PP i with data from the kinetics of PP i hydrolysis, PP i synthesis, and P i /HOH oxygen exchange. .…”

“…E. coli PPase requires four Mg 2ϩ ions per active site for catalysis, as described in Scheme I. This scheme, which fully accounts for the overall catalysis of PP i :P i equilibration by E. coli PPase, is a slightly modified version of the one proposed by Baykov et al (1990) following the approach developed for S. cerevisiae PPase (Springs et al, 1981;Welsh et al, 1983).…”

Oxygen exchange reactions catalyzed by vacuolar H+‐translocating pyrophosphatase Evidence for reversible formation of enzyme‐bound pyrophosphate

“…Pc is therefore a measure of the capacity of the V-PPase for E-P-P formation and thence reversal through the exclusion of water. (ii) The independence of the value of Pc from P~ concentration implies that of the two Pi molecules involved, the one containing the electrophilic center for attack by water is released from the complex first [26]. (iii) The exchange reaction cannot be limited by the rate of release of the second P~ into solution [12,26].…”

“…(ii) The independence of the value of Pc from P~ concentration implies that of the two Pi molecules involved, the one containing the electrophilic center for attack by water is released from the complex first [26]. (iii) The exchange reaction cannot be limited by the rate of release of the second P~ into solution [12,26]. Modulation of medium exchange by monovalent cations during the substrate hydrolytic cycle must necessarily correspond to PP~ hydrolysis and/or release of the first P~ molecule.…”