PMID:10739481
| Citation |
Avaeva, SM, Vorobyeva, NN, Kurilova, SA, Nazarova, TI, Polyakov, KM, Rodina, EV and Samygina, VR (2000) Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase. Biochemistry Mosc. 65:373-87 |
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| Abstract |
The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated. The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed. It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+. Different properties of these cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition. This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi. It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site. As a result, Ca2+ is a powerful inhibitor of all known PPases. Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered. |
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| Keywords |
Alanine/metabolism; Amino Acid Substitution; Asparagine/metabolism; Aspartic Acid/metabolism; Binding Sites; Calcium/pharmacology; Dialysis; Diphosphates/metabolism; Escherichia coli/enzymology; Glutamic Acid/metabolism; Hydrolysis; Inorganic Pyrophosphatase; Kinetics; Lanthanum/chemistry; Magnesium Compounds/metabolism; Models, Molecular; Pyrophosphatases/antagonists & inhibitors; Pyrophosphatases/genetics; Pyrophosphatases/metabolism |
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