Segmental motions of rat thymidylate synthase leading to half‐the‐sites behavior

Abstract

Thymidylate synthase (TS) is a homodimeric enzyme with two equivalent active sites composed of residues from both subunits. Despite the structural symmetry of the enzyme, certain experimental results are consistent with half-the-sites activity, suggesting negative cooperativity between the active sites. To gain insight into the mechanism behind this phenomenon, we explore segmental motions of rat TS in the absence of ligands, with normal mode analysis as a tool. Using solvent accessible surface area of the active site pocket as a monitor of the degree of opening of the active sites, we classified the first 25 nontrivial normal modes, obtained from the web server of the program ElNémo, according to the behavior of the active sites. We found seven modes that open and close both sites symmetrically and nine that do so in an anticorrelated fashion. We characterized the motions of these modes by visual inspection and through measurement of distances between selected atoms lining the active site pockets. The segments that regulate access to the active site correspond to the loop containing R44, helix K, and a long loop containing residues 103-125, in agreement with a large body of crystallographic studies. These elements can be activated together or in isolation. There are more asymmetric modes than symmetric ones in the set we analyzed, probably accounting for the half-the-sites behavior of the enzyme. Three of the asymmetric modes result in changes at the dimer interface and indicate the endpoints of possible communication pathways between the active sites.

Chemical shift imprint of intersubunit communication in a symmetric homodimer

“…Although dUMP was recently shown to bind with minimal cooperativity for the E. coli enzyme at 25°C, there are signs of unequal thermodynamics between the two protomers at lower temperatures (16), and indeed data herein show clear intersubunit communication. Moreover, other TS enzymes, and in particular human, seem to show more dramatic cooperativity, suggesting that intersubunit communication is an intrinsic feature of TS (13,(17)(18)(19)(20)(21). To overcome the difficulties of studying symmetric proteins by NMR, we generated a pair of mixed labeled dimers of TS that each have a single functional active site and a single protomer labeled for NMR studies.…”

Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-hydroxy-dCMP

“…4, lanes 6 and 12). Importantly, a consequence of TS being a half-the-sites reactive enzyme is that catalytic complex formation on one monomer renders the other monomer inactive, [35][36][37] with respect to both ligand binding and catalysis. Thus more frequent (if not exclusive) phosphorylation of one subunit appears to influence the latter mechanism by introducing subunit asymmetry and causing differing ligand binding and reactivity.…”

“…34 Thus differing interactions are suggested by the inhibitor with two binding sites on the enzyme molecule that may be interpreted in terms of negative cooperativity. In this context, it should be mentioned that although TS is a homodimer with two equivalent active sites, each composed of residues from both subunits, it shows half-the-site activity, with associated negative cooperativity, [35][36][37] the latter questioned recently to occur with the bacterial enzyme. 38 However, as presented in Table 1, the enzyme isolated from certain sources showed linear dependence of rate of inactivation by FdUMP on time, and both behaviors could be found in enzyme preparations isolated from sources of the same specific origin.…”

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

“…increase is unchanged after dUMP binding and is unique for Trp103; (2) dUMP binding provokes inhomogenous distributions of E.P. between subunits which could be a result of cooperation between them (mTS is known to exhibit negative cooperativity, thus being a half-the-sites active enzyme) [38][39][40]; (3) possibly a strong cooperation between Trp84 and Trp103, which at the closest point are approx. 5 Å away, exists.…”