Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase

Abstract

The structures of thymidylate synthase (TS) from Escherichia coli, in ternary complexes with substrate and an analogue of the cofactor, are the basis of a stereochemical model for a key reaction intermediate in the catalyzed reaction. This model is used to compare the reaction chemistry and chirality of the transferred methyl group with structures of the components, to identify those residues that participate, and to propose a stereochemical mechanism for catalysis by TS. Effects of chemical modification of specific amino acid residues and site-directed mutations of residues are correlated with structure and effects on enzyme mechanism. The ordered binding sequence of substrate deoxyuridine monophosphate and methylenetetrahydrofolate can be understood from the structure, where each forms a large part of the binding site for the other. The catalytic site serves to orient the reactants, which are sequestered along with many water molecules within a cavernous active center. Conformational changes during the reaction could involve nearby residues in ways that are not obvious in this complex.

The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism

“…The results show no measurable difference between the two mechanism when substituting 16 O4 oxygen by its 18 O4 isotope, but different trends are obtained on KIEs for 12 C6 to 13 C6 substitution when computed between reactants and TS in concerted and step-wise mechanism: a normal effect in the concerted mechanism and no effect in the step-wise one. As observed in Figure 6, a larger change in hybridization on C6 from reactants to TS is observed for the concerted mechanism (Figure 6B) than in the step-wise mechanism (Figure 6A) since the C6-S bond is elongated in the former and no change is detected in the step-wise TS.…”

“…It is important to mention that without tunneling correction the predicted KIEs are probably deflated (as in ref 48 ), but their trend is likely to be correct. Finally, heavy atoms KIEs, computed for 12 C to 13 C substitution at C6 position predict a normal effect (KIE=1.011 at AM1/MM and 1.0108 at M06-2X/MM) in the concerted mechanism and no effect (KIE=1) in the step-wise one. This result is not surprising as a change in hybridization has been observed from reactants to the concerted TS (the C6-S is slightly elongated in the TS), while no change has suffered C6 atom in the step-wise TS.…”

Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

“…The TS dimer is a half-site reactivity enzyme (at any given time, only one-half of the TS dimer is catalytically active) and conformational changes upon ligand binding lead to asymmetric binding sites with differential substrate affinities at each half of the dimer (22)(23)(24). We sought to determine whether coordination of the two TS active sites to provide half-site reactivity impacts DHFR activity: DHFR single enzyme turnover rates were determined under conditions where one or both TS active sites are predicted to be ligand-bound.…”

Mechanistic interpretation of conventional Michaelis–Menten parameters in a transporter system

“…For example, these include the ternary complex mechanism for glutathione S-transferases (3), the ping-pong mechanism for thioredoxin peroxidase (4), the random-sequential mechanism for IκB kinases α and β (5), and the ordered-sequential mechanism for thymidylate synthase (6). These models have benefited from a large number of experimental methods to study enzyme structure and function, resulting in models that are more elaborate than the Michaelis-Menten model.…”