A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli
| Autor: | Andriana P. Nikolova, Sirui Cao, David Satzer, Elizabeth E. Trimmer, Chong Zuo, David P. Ballou, Amber L. Jolly, Jeremy S. Sanchez |
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| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular 0301 basic medicine Half-reaction Protein Conformation NADH binding Glutamine Biophysics Flavoprotein Oxidative phosphorylation Flavin group Reductase Crystallography X-Ray Biochemistry Catalysis Substrate Specificity 03 medical and health sciences Folic Acid Escherichia coli Enzyme kinetics Molecular Biology Methylenetetrahydrofolate Reductase (NADPH2) chemistry.chemical_classification 030102 biochemistry & molecular biology biology Chemistry Escherichia coli Proteins NAD Kinetics Enzyme biology.protein Oxidation-Reduction |
| Zdroj: | Archives of Biochemistry and Biophysics. 642:63-74 |
| ISSN: | 0003-9861 |
| Popis: | The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6–10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme. |
| Databáze: | OpenAIRE |
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