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Entropy in Bi-substrate Enzymes: Proposed Role of an Alternate Site in Chaperoning Substrate into, and Products out of, Thymidylate Synthase

Authors
Journal
Journal of Molecular Biology
0022-2836
Publisher
Elsevier
Publication Date
Volume
255
Issue
3
Identifiers
DOI: 10.1006/jmbi.1996.0043
Keywords
  • Thymidylate Synthase
  • X-Ray Crystallography
  • Enzyme
  • Catalysis
Disciplines
  • Biology

Abstract

Abstract Three steps along the pathway of binding, orientation of substrates and release of products are revealed by X-ray crystallographic structures of ternary complexes of the wild-type Lactobacillus caseithymidylate synthase enzyme. Each complex was formed by diffusion of either the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate or the folate analog 10-propargyl- 5,8-dideazafolate into binary co-crystals of thymidylate synthase with 2′-deoxyuridine-5′-monophosphate. A two-substrate/enzyme complex is formed where the substrates remain unaltered. The imidazolidine ring is unopened and the pterin of the 5,10-methylene-5,6,7,8-tetrahydrofolate cofactor binds at an unproductive "alternate" site. We propose that the presence of the pterin at this site may represent an initial interaction with the enzyme that precedes all catalytic events. The structure of the 2′-deoxyuridine-5′-monophosphate and 10-propargyl-5,8-dideazafolate folate analog complex identifies both ligands in orientations favorable for the initiation of catalysis and resembles the productive complex. A product complex where the ligands have been converted into products of the thymidylate synthase reaction within the crystal, 2′-deoxythymidine-5′- monophosphate and 7,8-dihydrofolate, shows how ligands are situated within the enzyme after catalysis and on the way to product release.

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