Evidence of a Catalytic Dyad in F420-Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis.

IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Alaa Aziz, Lindsay A Davis, Ravi Ramkissoon, Neema Zeighami, Mansi Lohtia, Jamariya A Howard, Edward N Baker, Ghader Bashiri, Kayunta L Johnson-Winters
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引用次数: 0

Abstract

F420-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using cofactor F420 as the hydride transfer acceptor. Our previous pH dependence studies suggested that E109 serves as an active site acid, donating a proton to the N-1 position of F420, while leaving the role of H40 unanswered, which was previously suggested to serve as the active site base. This work utilizes thermodynamic and kinetic studies to elucidate additional mechanistic details concerning the roles of H40 and E13. The E13 residue had not previously been considered as a key player during catalysis. Therefore, the H40A, H40Q, E13A, and E13Q FGD variants were generated and fully characterized to determine their roles in catalysis. Here, we conducted temperature-dependent pH profiles and inactivation experiments using diethylpyrocarbonate (DEPC) to determine the role of H40 during catalysis. The temperature-dependent experiments suggest that an acidic histidine can donate a proton to E13. The inactivation experiments revealed monophasic kinetics, suggesting that the one active site H40 is covalently modified by DEPC. Therefore, the active site base is a deprotonated H40 that abstracts a proton from G6P, and then a hydride is transferred to the C-5 position of cofactor F420. These data suggest that E13 and H40 act as a catalytic dyad. Global analysis of the pre-steady-state experiments revealed the accumulation of an intermediate, the spectrum of which resembles an enzyme-product complex. The global analysis also reveals fast chemistry and slow product release with cofactor association being rate-limiting in catalysis.

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来源期刊
Biochemistry Biochemistry
Biochemistry Biochemistry 生物-生化与分子生物学
CiteScore
5.50
自引率
3.40%
发文量
336
审稿时长
1-2 weeks
期刊介绍: Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.
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