Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

Burçe Ergel,Michelle L Gill,Lewis Brown,Bomina Yu,Arthur G Palmer,John F Hunt

doi:10.1074/jbc.m114.575647

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.

Highlights

AlkB is an iron/2-oxoglutarate dioxygenase that repairs alkylated DNA
Calorimetric studies demonstrate substantial enthalpy-entropy compensation [60, 61] upon binding the metal cofactor and the 2OG co-substrate,5 suggesting the occurrence of a significant change in solvent interaction or protein dynamics even if there is not a substantial change in net secondary structure
By combining fluorescence spectroscopy and NMR spectroscopy with enzymological assays, we demonstrate that a specific protein conformational transition orchestrates the complex multistep catalytic reaction cycle of AlkB (Fig. 1D), controlling both the proper sequential order of cofactor/substrate binding as well as the kinetic sequestration of the reactive oxyferryl intermediate

Summary

Background

AlkB is an iron/2-oxoglutarate dioxygenase that repairs alkylated DNA. Results: Enzymological and biophysical methods are used to develop a quantitative mechanistic scheme. Extensive kinetic and spectroscopic studies have demonstrated that Fe(II)/2OG dioxygenases face a series of mechanistic challenges in achieving efficient catalysis [15,16,17,18,19,20,21,22] These enzymes catalyze similar multistep reactions in which the Fe(II) cofactor and 2OG co-substrate activate O2 for hydroxylation of a bioorganic substrate, an alkylated nucleobase in the case of AlkB [12, 13]. This so-called “primary substrate” occludes the binding sites for Fe(II) and 2OG in available crystal structures (as shown for AlkB in Fig. 1B [23]), consistent with the conclusion from kinetic studies (24 –26) that the enzymes bind the various cofactor and substrates sequentially, with the primary substrate binding only after both Fe(II) and 2OG (Fig. 1A). This long lived oxyferryl intermediate must remain tightly sequestered in the active site to avoid reaction with oxidizable compounds in the buffer

The abbreviations used are

EXPERIMENTAL PROCEDURES

57 Ϯ 14 74 Ϯ 19 90 Ϯ 5

RESULTS

DISCUSSION