文摘
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 kinact and KI 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 (kinact = 0.0058 ± 0.0005 min−1, KI = 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 kcat for the glutaminase activity 78-fold. Our findings suggest that the conformational change associated with binding ATP may be transmitted through the L10-α11 structural unit (residues 297–312) and thereby mediate effects on the glutaminase activity of CTPS.
