Details

Autor(en) / Beteiligte

• Johnson‐Winters, Kayunta
• Oyugi, Mercy
• Davis, Lindsay
• Bashiri, Ghader
• Baker, Edward N.

Titel

Determining the active site base and order of substrate addition within F 420 ‐dependent glucose‐6‐phosphate using steady‐state and pre steady‐state kinetics and isotope effects methods

Ist Teil von

• The FASEB journal, 2018-04, Vol.32 (S1)

Erscheinungsjahr

2018

Link zum Volltext

Quelle

Wiley Online Library

Beschreibungen/Notizen

• Abstract only F 420 ‐dependent glucose‐6‐phosphate dehydrogenase (FGD) is a Mycobacterial enzyme that catalyzes the conversion of glucose‐6‐phosphate (G6P) to 6‐phosphogluconolactone using oxidized F 420 . This enzyme is important within Mycobacteria tuberculosis , which is the causative agent of tuberculosis disease. Our work has focused on probing the FGD reaction mechanism using kinetic and isotope effects methods. Based upon a previous crystal structure FGD, there were two conserved amino acids, His40 and Glu109, which were we believed to function as the active site base and acid, respectively. Based upon pH profiles, we have determined that while Glu109 does serve as the active site acid, H40 does not serve as the active site base. We have identified several other amino acid candidates that could act as the active site base and will be discussed. A concurrent goal was to probe the FGD reaction mechanism using dead‐end inhibition experiments, along with solvent and substrate deuterium isotope effects studies. The dead‐end inhibition studies were conducted using the competitive inhibitor, citrate. The data revealed competitive and uncompetitive inhibition patterns for G6P and F 420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F 420 cofactor must first bind to FGD before G6P binding. Our proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed solvent kinetic isotope effect is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P‐d 1 yielded kinetic isotope effect (KIE) of 1.1 for both k cat and k cat / K m , while the pre‐steady state KIE on k obs was 1.4. Because the hydride transferred to C5 of F 420 was targeted for isotopic substitution, these KIE values provide further evidence to support our findings that hydride transfer is likely not rate‐limiting in the FGD reaction. Support or Funding Information 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.) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .