Mechanism of Ca²⁺-Induced Inhibition of Escherichia coli Inorganic Pyrophosphatase

S. M. Avaeva^1*, N. N. Vorobyeva², S. A. Kurilova¹, T. I. Nazarova¹, K. M. Polyakov³, E. V. Rodina¹, and V. R. Samygina³

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181; E-mail: avaeva@libro.genebee.msu.su

²School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 932-8846; E-mail: protein@libro.genebee.msu.su

³Shubnikov Institute of Crystallography, Leninskii pr. 59, Moscow, 117333 Russia; fax: (095) 135-1011; E-mail: kostya@ns.crys.ras.ru

^* To whom correspondence should be addressed.

Received October 11, 1999

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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 MgPP_i and LaPP_i 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 CaPP_i 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 PP_i. It was also demonstrated that Ca²⁺, as a constituent of the non-hydrolyzable substrate analog CaPP_i, competes with MgPP_i 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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KEY WORDS: inorganic pyrophosphatase, equilibrium dialysis, Ca²⁺, inhibition, complex, spatial structure

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