Allosteric regulation of yeast inorganic pyrophosphatase by substrate.
Kinetic and binding studies of yeast inorganic pyrophosphatase (EC 3.6.1.1) revealed a regulatory PPi-binding site. Rate vs substrate concentration dependencies were markedly nonhyperbolic in the range of 0.1-150 microM MgPPi at fixed Mg2+ levels of 0.05-10 mM provided that the enzyme had been preequilibrated with Mg2+. Imidodiphosphate, hydroxymethylenebisphosphonate, and phosphate eliminated the deviations from the Michaelis-Menten kinetics and inhibited PPi hydrolysis in a manner consistent with their binding at both active and regulatory sites. The results agreed with a model in which binding of uncomplexed PPi at the regulatory site markedly increases enzyme affinity for the activating Mg2+ ion. Ultrafiltration studies revealed the binding of at least 3 mol of the inhibitory hydroxymethylenebisphosphonate and of 2 mol of noninhibitory methylenebisphosphonate per mole of the dimeric enzyme.
Baykov
A A
AA
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Pavlov
A R
AR
Kasho
V N
VN
Avaeva
S M
SM
D000494:Allosteric Regulation; D001665:Binding Sites; D004164:Diphosphonates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D010710:Phosphates / Q000494:pharmacology; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004164:Diphosphonates; D010710:Phosphates; C029285:hydroxymethanediphosphonic acid; C016514:imidodiphosphonic acid; D011755:Pyrophosphatases; D008274:Magnesium