Alaa Aziz / Chemistry & Biochemistry / Faculty Mentor: Kayunta Johnson-Winters
F420- dependent glucose-6-phosphate dehydrogenase (FGD) is an important enzyme found in Mycobacteria tuberculosis, the causative agent of tuberculosis disease. FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using F420 cofactor as a hydride transfer acceptor. Understanding the FGD mechanism is vital to the design of new drugs for the treatment of multiple drug resistant and extreme drug resistant forms of tuberculosis disease. To this end, we have created a series of active site variants (H40A, H40Q, H260A , H260N, Glu13A, Glu13Q and Glu109A) and obtained the substrate binding affinities, determined the pre steady-state and steady state kinetic parameters and studied the order of substrate addition along with the pH- rate profiles for wtFGD and FGD variants. The binding studies suggest that these conserved amino acids are involved F420 binding but are not involved in G6P binding. The steady-state experiments suggest that the studied residues are important in catalysis due to decreased catalytic activity. Based upon the inhibition studies we have determined that FGD follows a sequential mechanism in which F420 binds first followed by G6P. The pH profiles along with the PROPKA calculations suggest that Glu13 and His40 function as a catalytic dyad, with His40 donating a proton to Glu13. His40 can then act as an active site base, abstracting a proton from G6P facilitating the reduction of the F420 cofactor. The global analysis of the pre steady-state kinetic data suggest that hydride transfer is not rate-limiting in catalysis, and that the mechanism follows a fast chemistry and slow product release with no observable intermediates
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