The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2',3'-dialdehyde ATP and mutational analyses.
Cytidine 5'-triphosphate synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or L-glutamine as the source of nitrogen. To identify the location of the ATP-binding site within the primary structure of E. coli CTPS, we used the affinity label 2',3'-dialdehyde adenosine 5'-triphosphate (oATP). oATP irreversibly inactivated CTPS in a first-order, time-dependent manner while ATP protected the enzyme from inactivation. In the presence of 10 mM UTP, the values of k(inact) and K(I) were 0.054 +/- 0.001 min(-1) and 3.36 +/- 0.02 mM, respectively. CTPS was labeled using (2,8-3H)oATP and subsequently subjected to trypsin-catalyzed proteolysis. The tryptic peptides were separated using reversed-phase HPLC, and two peptides were identified using N-terminal sequencing (S(492)GDDQLVEIIEVPNH(506) and Y(298)IELPDAY(K(306)) in a 5:1 ratio). The latter suggested that Lys 306 had been modified by oATP. Replacement of Lys 306 by alanine reduced the rate of oATP-dependent inactivation (k(inact) = 0.0058 +/- 0.0005 min(-1), K(I) = 3.7 +/- 1.3 mM) and reduced the apparent affinity of CTPS for both ATP and UTP by approximately 2-fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation was also reduced 2-fold while near wild-type activity was observed when NH3 was the substrate. These findings suggest that Lys 306 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between the ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain. Replacement of the nearby, fully conserved Lys 297 by alanine did not affect NH3-dependent CTP formation, relative to wild-type CTPS, but reduced k(cat) for the glutaminase activity 78-fold. Our findings suggest that the conformational change associated with binding ATP may be transmitted through the L10-alpha11 structural unit (residues 297-312) and thereby mediate effects on the glutaminase activity of CTPS.[1]References
- The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2',3'-dialdehyde ATP and mutational analyses. MacLeod, T.J., Lunn, F.A., Bearne, S.L. Biochim. Biophys. Acta (2006) [Pubmed]
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