title: Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase.
abstract: Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5-20 mM free Mg2+ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 +/- 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg2+. The Pi-inhibition pattern of pyrophosphate hydrolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi-binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.
authors.affiliation: A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, U.S.S.R.
authors.forename: I N
authors.initials: IN
authors.lastname: Smirnova
authors.forename: A S
authors.initials: AS
authors.lastname: Shestakov
authors.forename: E B
authors.initials: EB
authors.lastname: Dubnova
authors.forename: A A
authors.initials: AA
authors.lastname: Baykov
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008962:Models, Theoretical; D010710:Phosphates / Q000494:pharmacology*; D011755:Pyrophosphatases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry
chemical_list: D010710:Phosphates; D011755:Pyrophosphatases; D008274:Magnesium