Details

Autor(en) / Beteiligte

• Oyugi, Mercy A
• Bashiri, Ghader
• Baker, Edward N
• Johnson-Winters, Kayunta

Titel

Investigating the Reaction Mechanism of F 420 -Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis: Kinetic Analysis of the Wild-Type and Mutant Enzymes

Ist Teil von

• Biochemistry (Easton), 2016-10, Vol.55 (39), p.5566-5577

Ort / Verlag

United States

Erscheinungsjahr

2016

Quelle

MEDLINE

Beschreibungen/Notizen

• F -dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using F cofactor 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 F , thus suggesting that Trp44, His40, and Glu109 aid in F binding. 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 k -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 F cofactor. 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.