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J. Biol. Chem., Vol. 283, Issue 4, 2010-2020, January 25, 2008

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Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax^*

From the Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH₃ or L-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH₃-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP 6-thio-GTP > ITP guanosine 5'-tetraphosphate > O⁶-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH₂ group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH₂ group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH₃- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC₅₀ 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC₅₀ 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC₅₀ = 80 µM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors.

Received for publication, September 18, 2007 , and in revised form, October 29, 2007.

^* This work was supported by the Canadian Institutes of Health Research (CIHR) and the CIHR Institute of Cancer Research through operating grants (to S. L. B.) and by graduate student fellowships from the Nova Scotia Health Research Foundation (to F. A. L. and J. E. M.), the Cancer Research Training Program (to F. A. L.), and the Walter C. Sumner Foundation (to F. A. L.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1S and Figs. 1S-4S.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada. Tel.: 902-494-1974; Fax: 902-494-1355; E-mail: sbearne{at}dal.ca.

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