X-ray structures of novel intermediates in the thymidylate synthase models for chemical mechanism and conformational change

Persistent Link:
http://hdl.handle.net/10150/279920
Title:
X-ray structures of novel intermediates in the thymidylate synthase models for chemical mechanism and conformational change
Author:
Arendall, William Bryan
Issue Date:
2001
Publisher:
The University of Arizona.
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.
Abstract:
The catalytic mechanism of thymidylate synthase (TS) was investigated using X-ray crystallography: four structures that yield new information about the early stages of TS action are reported. TS catalyzes the production of thymidylate (TMP), one of the four nucleotide bases of DNA, from the substrate, deoxyuridylate and cofactor, methylenetetrahydrofolate (MTF). Knowledge about the TS mechanism is important for both the medical and basic sciences. TS is the sole de novo source of TMP and it is thus a target for anti-proliferative drugs aimed at addressing cancer and other diseases marked by rapidly dividing cells. To aid this effort, past research on TS has developed two models to explain how TS works. A detailed, sequential chemical mechanism explains the methylene and hydride transfers from one cofactor to the substrate. And, a two state, dynamical model explains the conformational change that TS undergoes during its catalytic cycle. Combining these two models will lead to a fuller understanding of protein structure, function, and dynamics interrelationships. Two of the new structures contain cofactor in a heretofore unseen state, bound in the active site with its imidazolidine ring intact. Finding that this is an allowed enzyme-cofactor state indicates that ring opening and formation of the highly reactive iminium cation may occur relatively late in the methylene transfer, after preparation of the substrate; and, the reaction may perhaps be concerted. Further, details of these two structures show that protonation of the correct imidazolidine ring nitrogen (N10) may be selected by the geometry and environment imposed on the bent cofactor by TS. N5, the "wrong" ring nitrogen, is blocked and in a hydrophobic environment, while N10 is rehybridized to sp3 and its lone pair (nascent hydrogen) is pointed into an aqueous cavity trapped within the enzyme. A proposal coming from this dissertation is for a combination of the two models describing TS catalysis. The chemical mechanism model and the conformational change model both describe the same phenomena and these models should be connected and combined into one larger model to further increase our knowledge of the connections between structure, dynamics and function. The four structures reported here begin that connection process.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemistry, Biochemistry.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry and Molecular Biophysics
Degree Grantor:
University of Arizona
Advisor:
Montfort, William R.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleX-ray structures of novel intermediates in the thymidylate synthase models for chemical mechanism and conformational changeen_US
dc.creatorArendall, William Bryanen_US
dc.contributor.authorArendall, William Bryanen_US
dc.date.issued2001en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractThe catalytic mechanism of thymidylate synthase (TS) was investigated using X-ray crystallography: four structures that yield new information about the early stages of TS action are reported. TS catalyzes the production of thymidylate (TMP), one of the four nucleotide bases of DNA, from the substrate, deoxyuridylate and cofactor, methylenetetrahydrofolate (MTF). Knowledge about the TS mechanism is important for both the medical and basic sciences. TS is the sole de novo source of TMP and it is thus a target for anti-proliferative drugs aimed at addressing cancer and other diseases marked by rapidly dividing cells. To aid this effort, past research on TS has developed two models to explain how TS works. A detailed, sequential chemical mechanism explains the methylene and hydride transfers from one cofactor to the substrate. And, a two state, dynamical model explains the conformational change that TS undergoes during its catalytic cycle. Combining these two models will lead to a fuller understanding of protein structure, function, and dynamics interrelationships. Two of the new structures contain cofactor in a heretofore unseen state, bound in the active site with its imidazolidine ring intact. Finding that this is an allowed enzyme-cofactor state indicates that ring opening and formation of the highly reactive iminium cation may occur relatively late in the methylene transfer, after preparation of the substrate; and, the reaction may perhaps be concerted. Further, details of these two structures show that protonation of the correct imidazolidine ring nitrogen (N10) may be selected by the geometry and environment imposed on the bent cofactor by TS. N5, the "wrong" ring nitrogen, is blocked and in a hydrophobic environment, while N10 is rehybridized to sp3 and its lone pair (nascent hydrogen) is pointed into an aqueous cavity trapped within the enzyme. A proposal coming from this dissertation is for a combination of the two models describing TS catalysis. The chemical mechanism model and the conformational change model both describe the same phenomena and these models should be connected and combined into one larger model to further increase our knowledge of the connections between structure, dynamics and function. The four structures reported here begin that connection process.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemistry, Biochemistry.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMontfort, William R.en_US
dc.identifier.proquest3002540en_US
dc.identifier.bibrecord.b41434237en_US
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