Mechanistic studies on F 420 ‐dependent glucose‐6‐phosphate dehydrogenase using kinetic isotope effect methods and pH profiles

Abstract

F420-dependent Glucose-6-phosphate Dehydrogenase (FGD) is 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 oxidized F420 cofactor, which becomes reduced during catalysis (Figure 1). Previous crystallographic studies on wild-type FGD suggested that conserved residues, H40 and Glu 109 acted as the active site base and active site acid, respectively. However, our previous pH profile experiments suggested that while Glu 109 does act as the acid, His 40 does not act as the active site base. Our present work focuses on determining which active site residue could act as the active site base. For this reason, we have created FGD variants of Glu13,, His 260 and H40. We have conducted kinetic isotope effect experiments as well as pH dependence studies in order to elucidate their roles during catalysis. Our results will be discussed here.