Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence

被引:48
作者
Baykov, AA
Hyytia, T
Volk, SE
Kasho, VN
Vener, AV
Goldman, A
Lahti, R
Cooperman, BS
机构
[1] UNIV PENN,DEPT CHEM,PHILADELPHIA,PA 19104
[2] UNIV CALIF LOS ANGELES,DEPT MED,CTR ULCER RES & EDUC,LOS ANGELES,CA 90073
[3] CTR BIOTECHNOL,SF-20521 TURKU,FINLAND
[4] UNIV TURKU,DEPT BIOCHEM,SF-20500 TURKU,FINLAND
关键词
D O I
10.1021/bi952635u
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphate (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measurements 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 pK(a)s of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+ >0.2 mM, The model straightforwardly accounts for the apparent linkage between increased pK(a) 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-P-i 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 P-i-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and P-i-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released P-i and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound P-i, respectively, as the pH is raised.
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收藏
页码:4655 / 4661
页数:7
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