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J. Biol. Chem., Vol. 282, Issue 10, 7242-7253, March 9, 2007

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Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria^*

Cécile Evrin, Monica Straut, Neli Slavova-Azmanova, Nadia Bucurenci, Adrian Onu, Liliane Assairi^¶, Mihaela Ionescu, Nicolae Palibroda^||, Octavian Bârzu, and Anne-Marie Gilles¹

From the UnitédeGénétique des Génomes Bactériens, Institut Pasteur, 75724 Paris Cedex, France, the Laboratory of Enzymology and Applied Microbiology, Cantacuzino Institute, 050096 Bucharest, Romania, ^¶INSERM U759, Institut Curie-Recherche, Centre Universitaire Paris-Sud, Bâtiments 110-112, 91405 Orsay, France, and the ^||Institute of Isotopic and Molecular Technology, 400293 Cluj-Napoca, Romania

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5–10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K_m for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.

Received for publication, July 21, 2006 , and in revised form, January 8, 2007.

^* This work was supported by CNRS Grants URA 2185 and URA 2171, Institut Pasteur Grant ACO2 and by AstraZeneca R&D Boston. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Unitéde Génétique des Génomes Bactériens, Inst. Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-4568-8968; Fax: 33-1-4568-8948; E-mail: amgilles{at}pasteur.fr.

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