Document Type
Honors Thesis
Abstract
F420-dependent glucose-6-phosphate dehydrogenase (FGD) is an essential enzyme found within Mycobacterium tuberculosis, the causative agent of tuberculosis disease (TB). FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using the unique F420 cofactor. This reaction is carried out as the first step in the pentose phosphate pathway. This current project will expand on previous work conducted on the H260N FGD variant, aimed at determining if Histidine 260 serves as an active site base. We have conducted fluorescence binding, steady-state, pH dependence profiles, and pre steady-state kinetic experiments on wild type FGD as well as the His260 FGD variant. The results from the pH profiles suggest that H260N does not serve as the active site base. However, it plays an important role in catalysis due to lack of activity of the H260N variant. The steady-state substrate kinetic isotope effects using G6P and G6P-d1 yielded a KIE of 0.95 while the pre steady state yielded a KIE value of 1.02.Based on this, the KIE values provide evidence that the hydride transfer is not the rate limiting step and His260 does not act as the active site base.
Publication Date
5-1-2019
Language
English
License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Monyancha, Diana, "CHARACTERIZATION OF F420 DEPENDENT GLUCOSE-6-PHOSPHATE DEHYDROGENASE H260N VARIANT USING STEADY STATE, PRESTEADY STATE AND KINETIC ISOTOPE EFFECT METHODS" (2019). 2019 Spring Honors Capstone Projects. 32.
https://mavmatrix.uta.edu/honors_spring2019/32