Investigating the Reaction Mechanism of F420-Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis: Kinetic Analysis of the Wild-Type and Mutant Enzymes.
F420-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using F420cofactor as the hydride transfer acceptor, within mycobacteria. A previous crystal structure of wild-type FGD led to a proposed mechanism suggesting that the active site residues His40, Trp44, and Glu109 could be involved in catalysis. We have characterized the wild-type FGD and five FGD variants (H40A, W44F, W44Y, W44A, and E109Q) by fluorescence binding assays and steady-state and pre-steady-state kinetic experiments. Compared to wild-type FGD, all the variants had lower binding affinities for F420, thus suggesting that Trp44, His40, and Glu109 aid in F420binding. While all the variants had decreased catalytic efficiencies, FGD H40A and W44A were the least efficient, having lost ∼1000- and ∼2000-fold activity, respectively. This confirms a crucial catalytic role for His40 in the FGD reaction and suggests that aromaticity at residue 44 aids catalysis. To investigate the proposed roles of Glu109 and His40 in acid-base catalysis, the pH dependence of kinetic parameters has been determined for the E109Q and H40A mutants and compared to those of the wild-type enzyme. The log kcat-pH profile of wild-type FGD and E109Q revealed two ionizable residues in the enzyme-substrate complex, while H40A displayed only one ionization event. The FGD E109Q variant displayed pH-dependent kinetic cooperativity with respect to the F420cofactor. The multiple-turnover pre-steady-state kinetics were biphasic for wild-type FGD, W44F, W44Y, and E109Q, while the H40A and W44A variants displayed only a single phase because of their reduced catalytic efficiency.
Department of Chemistry and Biochemistry, The University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
Mercy A
MA
Oyugi
Laboratory of Structural Biology and Maurice Wilkins Center for Molecular Biodiscovery, School of Biological Sciences, The University of Auckland , Auckland 1010, New Zealand.
Ghader
G
Bashiri
Laboratory of Structural Biology and Maurice Wilkins Center for Molecular Biodiscovery, School of Biological Sciences, The University of Auckland , Auckland 1010, New Zealand.
Edward N
EN
Baker
Department of Chemistry and Biochemistry, The University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
Kayunta
K
Johnson-Winters
D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005954:Glucosephosphate Dehydrogenase