Thymidylate synthase (TSase) is an enzyme that catalyzes the conversion of uridylate to thymidylate, one of the four DNA building blocks. Functional perturbation of this enzyme causes thymidylate deprivation in cells, thereby halting DNA production. This makes TSase crucial for cell survival and proliferation. TSase is an attractive target for chemotherapy as treatment for colorectal, ovarian, pancreatic and an array of other cancers. TSase is one of the most highly conserved enzymes across the whole spectrum of life. There is more than 45% sequence identity between E. coli and human TSases. Similarities are also observed in three-dimensional structures, functions and other kinetic and mechanistic features among most TSases from very diverse origins. Thereby, E. coli TSase is used as a model system to investigate the properties of its human counterpart.
In TSase catalysis, two H-transfers are involved: a proton abstraction and a hydride transfer. Traditional experimental biochemical kinetic studies and, more recently, computational studies (QM/MM) have been used to probe the mechanism of both H-transfers in E. coli TSase. While the traditional mechanism proposes a step-wise process for the hydride transfer, the QM/MM studies predict a concerted mechanism. The computational studies offered refined insight into the mechanism of proton abstraction by postulating the existence a novel, not covalently bound intermediate comprised of both ligands of the enzyme. Our mutational, kinetic and isotope effect work indeed supports those alternative mechanisms for the hydride transfer and the proton abstraction. The novel intermediate, which was synthesized and confirmed to be kinetically competent, opens a new avenue for finding a new class of TSase inhibitors.
Despite very high similarities in the structure and the function between E. coli and human TSase, the latter one possesses some distinctive features such as an N-terminal tail, two insertions of 8-12 residues and active-inactive conformations. Finding other functional differences between bacterial and mammalian TSase could pave the way for designing species-specific inhibitors. Our kinetic and mechanistic studies reveal some unique characteristics of human TSase, such as insensitivity of the rate to Mg2+, a more random binding sequence of ligands and a somewhat different environment of the transition state for the hydride transfer and the proton abstraction. These characteristic properties of human TSase might have implications in designing inhibitors of bacterial TSase.
5-FU is a common chemotherapeutic drug that targets TSase and has been used for the treatment of a wide variety of cancers. However, it was reported that a phenotype of colorectal cancerous cells in a certain group of patients had differential sensitivity to 5-FU drug treatment. Though the overexpression of TSase protein, its mRNA and folate receptors has previously been implicated in drug resistance, this phenotype contained a regular level of TSase. Analysis of mRNA and its cDNA revealed a single base mutation in the TSase gene, which corresponded to a point mutation of Y33 to H in the TSase polypeptide chain. The residue Y33 is remotely located and has no direct contact with either of the substrates; nevertheless, the mutation confers resistance to the drug. We aimed to obtain a comprehensive understating behind the resistance profile of Y33H. We prepared the recombinant Y33H protein and checked its inhibition profile. We found that the recombinant Y33H also confers resistance, like the one isolated from the resistant colorectal cancerous cells. There is a three-fold difference in the turnover number between the WT and the mutant, and our isotope effect experiments reveal altered transition state structures for the hydride transfer in the mutant enzyme. A crystal structure of an E. coli equivalent mutant is found perfectly superimposable on the WT human TSase structure, suggesting that no dramatic structural alteration is likely to to be present to account for the drug resistance and other changed behavior for the variant enzyme. However, analyses of isotropic B-factors of E. coli equivalent mutant and WT human TSase suggest alteration in protein dynamics across the protein chain, which imparts flexibility to the protein. Thermal melting temperature analyses of both the human WT and the mutant Y33H corroborates the notion of increased flexibility obtained from the dynamic features of crystal structures. Thereby, it seems that an alteration in protein dynamics compromises the drug sensitivity of the phenotype of cells harboring the Y33H mutant.