Open AccessKinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions
Author(s) -
BAYKOV Alexander A.,
SHESTAKOV Alexander S.,
KASHO Vladimir N.,
VENER Alexander V.,
IVANOV Artemiy H.
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb19482.x
Subject(s) - pyrophosphate , inorganic pyrophosphatase , chemistry , pyrophosphatase , hydrolysis , catalysis , reaction rate constant , substrate (aquarium) , enzyme kinetics , enzyme , stereochemistry , kinetics , inorganic chemistry , active site , organic chemistry , physics , oceanography , quantum mechanics , geology
Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme‐bound pyrophosphate formation and Mg 2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg 2+ . We determined the equilibrium constants for Mg 2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PP i , hydrolysis/synthesis of PP i and successive binding/release of two P i molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg 2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg 2+ is increased by two orders of magnitude in the enzyme‐substrate complexes. The distribution of 18 O‐labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme‐bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.