Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase.
Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme-bound and free pyrophosphate were measured over wide ranges of Mg2+ and respective substrate concentrations. Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange [Kasho, V. N. & Baykov, A. A. (1989) Biochem. Biophys. Res. Comm. 161, 475-480], yielded values of the equilibrium constants for Mg2+ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi bound. Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four-metal pathway. In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four-metal pathway, dissociation of the second Pi is faster for the three-metal pathway, while PPi hydrolysis and the release of the first Pi may proceed with similar rates. Release of pyrophosphate formed on the enzyme is faster for the three-metal pathway. Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg2+ concentration, thus providing a means for pyrophosphatase-activity regulation.
Baykov
A A
AA
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Shestakov
A S
AS
D001665:Binding Sites; D002384:Catalysis; D011756:Diphosphates / Q000378:metabolism*; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D017616:Magnesium Compounds; D011755:Pyrophosphatases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013816:Thermodynamics
D011756:Diphosphates; D017616:Magnesium Compounds; C034732:magnesium pyrophosphate; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium