Date of Award

8-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Nina Goodey

Committee Member

Jim Dyer

Committee Member

David Konas

Subject(s)

Tetrahydrofolate dehydrogenase--Inhibitors--Specifications

Abstract

Dihydrofolate reductase (DHFR) is an essential enzyme necessary in the synthesis of DNA precursors. DHFR acquires resistance to numerous known anti-folates and this has led to the continuous need to discover novel and selective DHFR inhibitors. Allosteric mutations on Bacillus stearothermophilus (Bs.) DHFR were previously found to alter inhibitor binding and specificity. In this work, we investigate how these mutations affect the conformational motions of DHFR associated with inhibitor binding. Two distal allosteric mutations, isoleucine at position 86 to alanine (I86A) and tyrosine at position 127 to alanine (Y127A) were separately introduced to C73A/S131C, a previously modified wild type DHFR to give C73A/I86A/S131C and C73A/Y127A/S131C DHFRs. The modified C73A/S131C DHFR contains a site for attachment of fluorescent label, N- [2-(l-maleimidyl) ethyl]-7-(diethyl amino) coumarin-3-carboxamide (MDCC). Development of C73A/S131Cmdcc DHFR allowed for a method to study two native conformations of DHFR that could otherwise not be studied by intrinsic fluorescence alone. Stopped-flow instrumentation together with fluorescence is used to study the kinetics of protein structural motions. This approach allowed us to examine the conformational changes associated with methotrexate (MTX) and pyrimethamine (PYR) binding, for the mutants and compare them to C73A/S131C DHFR. Intrinsic tryptophan fluorescence of the three DHFR constructs (C73A/S131C, 186A, Y127A) revealed a different conformational change upon PYR binding to one conformer compared to MTX binding. To study the second conformer, MDCC is covalently attached to the cysteine at position 131 on the DHFR (C73A/S131Cmdcc» I86Amdcc? Y127Amdcc) and all three enzymes show a conformational change, different from that observed by intrinsic fluorescence upon inhibitor binding that are inhibitor specific. This study provides insight on the conformational changes associated with inhibitor binding. How the conformational changes are inhibitor specific. As well as how the mutants’ (I86A and Y127A) affect the conformational changes compared to C73A/S131C. Understanding what happens during inhibitor binding and the role of allosteric mutations on ligand binding specificity can inform design and repurposing of selective DHFR drug compounds.

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