Abstract 1821 Investigating the Catalytic Mechanism of F420-dependent Glucose-6-phosphate Dehydrogenase Through Kinetic and Thermodynamic Analyses

F420-dependent glucose-6-phosphate dehydrogenase (FGD) is an enzyme found in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease. FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, utilizing an F420 cofactor as a hydride acceptor. This reaction is important in the production of the reduced F420 cofactor and nucleic acid biosynthesis within Mtb, as well as the development of pharmaceuticals to treat multi-drug resistant and extreme drug resistant forms of tuberculosis. Our goal is to understand the reaction mechanism of FGD. An initial proposed mechanism suggests that H40 acted as an active site base, while E109 functioned as the active site acid. Our previous studies confirmed that while E109 does act as the active site acid, H40 does not act as the active site base. To further our investigation, we have generated a series of FGD variants, which include E13A, E13Q, E109A, H40A, H40Q, H260A, H260N to further study H40, while determining the functionality of E13 and H260. We then obtained the binding affinities, steady-state, pre-steady-state kinetic parameters, and pH-rate profiles for wtFGD and the above mentioned FGD variants. The dissociation constant values showed that these amino acids aided in F420 binding but not G6P binding. The steady-state experiments revealed that the residues were important in catalysis due to decreased catalytic activity. The pKa of ionizable groups identified from pH-rate profiles were also investigated in wtFGD as a function of temperature and linearized to the van't Hoff equation to determine standard enthalpy and entropy of active site residue ionization. The results are discussed here. This work was supported by NIH Grant 1R15GM113223-01A1 (to KJW) and the Health Research Council of New Zealand Grant HRC 12/1111 (to E.N.B.).