Metal-Free PP_i Activates Hydrolysis of MgPP_i by an Escherichia coli Inorganic Pyrophosphatase

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Ju. P. Vainonen¹, N. N. Vorobyeva¹, E. V. Rodina^1,2*, T. I. Nazarova², S. A. Kurilova², Ju. S. Skoblov³, and S. M. Avaeva²

¹Chemistry Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: rodina@belozersky.msu.ru

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: nazarova@belozersky.msu.ru

³Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, 119991 Moscow, Russia; fax: (7-095) 135-1405; E-mail: skoblov@eimb.ru

^* To whom correspondence should be addressed.

Received March 19, 2004; Revision received April 21, 2004

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Soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) is a hexamer forming under acidic conditions the active trimers. We have earlier found that the hydrolysis of a substrate (MgPP_i) by the trimers as well as a mutant E-PPase Asp26Ala did not obey the Michaelis-Menten equation. To explain this fact, a model has been proposed implying the existence of, aside from an active site, an effector site that can bind PP_i and thus accelerate MgPP_i hydrolysis. In this paper, we demonstrate that the noncompetitive activation of MgPP_i hydrolysis by metal-free PP_i can also explain kinetic features of hexameric forms of both the native enzyme and the specially obtained mutant E-PPase with a substituted residue Glu145 in a flexible loop 144-149. Aside from PP_i, its non-hydrolyzable analog methylene diphosphonate can also occupy the effector site resulting in the acceleration of the substrate hydrolysis. Our finding that two moles of [³²P]PP_i can bind with each enzyme subunit is direct evidence for the existence of the effector site in the native E-PPase.

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KEY WORDS: inorganic pyrophosphatase, hexamer, trimer, effector site, substrate-induced activation, Glu145Gln substitution

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