Siqi Du

Graduation Semester and Year

2015

Language

English

Document Type

Thesis

Degree Name

Master of Science in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Kayunta Johnson-Winters

Abstract

F420 cofactor, a flavin analogue, is a unique two electron carrier that has been found in a number of microorganisms. This thesis focuses on two F420-dependent enzymes (F420-dependent glucose-6-phosphate dehydrogenase and F420H2: NADP+ Oxidoreductase). First, F420-dependent glucose-6-phosphate dehydrogenase (FGD) from Mycobacterium tuberculosis has been shown to be essential for activation of the anti-TB prodrug PA-824. It has been suggested the Trp44 residue in the active side aids in the stabilization of possible intermediates during catalysis. Here, we examined the role of Trp44 during catalysis by creating the FGD W44A variant, which was catalytically inactive. However, we were able to isolate the W44A variant through gel filtration methods and then conduct binding studies. The results suggest that Trp44 aids in the binding of F420 cofactor to FGD, but not glucose-6-phosphate binding. F420H2: NADP+ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP+, producing NADPH via a hydride transfer from the reduced F420 cofactor. The focus of the Fno studies is to elucidate the reaction mechanism of Fno and to assess the probability of hydrogen tunneling within the enzyne. Kinetic isotope effect methods has been employed to examine the chemical steps of this enzymatic reaction. The results suggest that the hydride transfer in not the rate-limiting step. However, evidence shows the solvent plays an important role during this catalyzed reaction. Furthermore, no evidence of hydrogen tunneling is seen in this investigation.

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

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