Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence.
Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM. The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised .
Baykov
A A
AA
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Hyytia
T
T
Volk
S E
SE
Kasho
V N
VN
Vener
A V
AV
Goldman
A
A
Lahti
R
R
Cooperman
B S
BS
D002384:Catalysis; D011756:Diphosphates / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D017616:Magnesium Compounds / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism*; D013379:Substrate Specificity
D011756:Diphosphates; D017616:Magnesium Compounds; C034732:magnesium pyrophosphate; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium