Bacterial versus human thymidylate synthase: Kinetics and functionality.

Zahidul Islam,Ilya Gurevic,Ananda K Ghosh,Timothy S Strutzenberg,Amnon Kohen,Tasnia Iqbal,Anna Roujeinikova

doi:10.1371/journal.pone.0196506

Abstract

Thymidylate Synthase (TSase) is a highly conserved enzyme that catalyzes the production of the DNA building block thymidylate. Structurally, functionally and mechanistically, bacterial and mammalian TSases share remarkable similarities. Because of this closeness, bacterial enzymes have long been used as model systems for human TSase. Furthermore, while TSase inhibitors have long served as chemotherapeutic drugs, no TSase inhibitor serves as an antibiotic. Despite their high resemblance, the mammalian TSases are distinct in a few known aspects, such as having a N-terminal tail and two insertions in the primary sequence and active/inactive conformations. Here, we aim to comprehensively characterize human (hs) TSase and delineate its contrasts and the similarities to the well-studied Escherichia coli (ec) TSase. We found that, in contrast to ecTSase, Mg2+ does not enhance reaction rates for hsTSase. The temperature dependence of intrinsic kinetic isotope effects (KIEs), on the other hand, suggests that Mg2+ has little or no impact on the transition state of hydride transfer in either enzyme, and that the transition state for the hydride transfer in hsTSase is looser than in ecTSase. Additionally, the substrates’ binding order is strictly ordered for ecTSase but slightly less ordered for hsTSase. The observed kinetic and functional differences between bacterial and human enzymes may aid in the development of antibiotic drugs with reduced toxicity.

Highlights

The last committed step in the de novo synthesis of thymidylate (2’-deoxythymidine-5’-monophosphate, dTMP) in most living organisms is catalyzed by the enzyme thymidylate synthase (TSase)
LB powder was purchased from Research Products International, Inc. [5-3H]-dUMP, specific radioactivity ~14 Ci/mmol and [2-14C]-dUMP, specific radioactivity ~53 mCi/ mmol, for hydride transfer experiments were from Moravek Biochemicals
methylene tetrahydrofolate (MTHF) substrate-inhibition is known for ecTSase because of an alternative unproductive binding mode of the cofactor [18, 34]

Summary

Introduction

The last committed step in the de novo synthesis of thymidylate (2’-deoxythymidine-5’-monophosphate, dTMP) in most living organisms is catalyzed by the enzyme thymidylate synthase (TSase). Suppression of TSase activity in cells causes a deficiency in the intracellular concentration of thymidylate, bringing DNA reproduction to a halt and causing death to cells [1]. The participation of TSase in the biosynthesis of DNA renders this enzyme a suitable target for anticancer drugs [2, 3]. Two well-known drugs, 5-fluorouracil and raltitrexed, that target.

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