Thymidylate synthase (TS) catalyzes the conversion of dUMP into dTMP via a methyl transfer from the cofactor, 5,10-methylenetetrahydrofolate (CH2THF), which is converted to dihydrofolate (DHF) during the reaction. Because this reaction is a step in the only de novo pathway leading to thymidine nucleotides, there has been considerable interest in TS inhibition for the treatment of proliferative disease. This had led to a large body of biochemical and structural data and a proposed reaction mechanism. Despite this extensive study, there are a number of unanswered questions regarding TS function. The role of the large ligand-induced conformational change is poorly understood, and many steps in the proposed mechanism of catalysis are unconfirmed. The roles of many evolutionarily conserved residues are also poorly understood, notably those located away from the active site. In this work, 23 structures of E. coli TS are presented. These structures are of eight site-specific mutants bound to several ligand combinations. The results of this work cast doubt on a number of aspects of previously published models of the reaction mechanism, and lead to a new proposed model. TS appears to use strain, electrostatic interactions, and conformational change to influence the stability, and thus reactivity, of the catalytic enzyme-substrate covalent bond. This work adds to a growing body of data suggesting that the stability of this bond is variable. Instability of this bond is central to the new proposed reaction mechanism. The mechanism proposed here accounts for the reaction without requiring a large isomerization of the ligand complex that was previously thought to be necessary. Also presented is evidence that CH2THF binds to TS without opening of the 5-membered ring and in a conformation similar to that of other TS-bound folates but different from the conformation observed in solution. The roles played by many conserved residues appears to be subtle, as evidenced by the small structural changes of mutants when compared to wild-type. The conservation of these residues suggests that TS is a highly optimized enzyme under strong selective pressure to maintain maximum catalytic activity.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/282542 |
| Date | January 1997 |
| Creators | Hyatt, David C., 1961- |
| Contributors | Montfort, William R. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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