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J Biol Chem. 2014 Oct 24;289(43):29584-601. doi: 10.1074/jbc.M114.575647. Epub 2014 Jul 20.

Protein dynamics control the progression and efficiency of the catalytic reaction cycle of the Escherichia coli DNA-repair enzyme AlkB.

Author information

1
From the Department of Biological Sciences, Columbia University, New York, New York 10027-6601 and.
2
the Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032-3702.
3
the Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032-3702 agp6@columbia.edu.
4
From the Department of Biological Sciences, Columbia University, New York, New York 10027-6601 and jfhunt@biology.columbia.edu.

Abstract

A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics.

KEYWORDS:

AlkB; Catalytic Efficiency; DNA Repair; Enzyme Mechanism; Fe-2OG Dioxygenase; Fluorescence Spectroscopy; Nuclear Magnetic Resonance (NMR); Protein Dynamic; X-ray Crystallography

PMID:
25043760
PMCID:
PMC4207975
DOI:
10.1074/jbc.M114.575647
[Indexed for MEDLINE]
Free PMC Article

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