NDLI: EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

Content Provider	PubMed Central
Author	Ruszczycky, Mark W. Choi, Sei-hyun Liu, Hung-wen
Abstract	The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, , when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of , yielded a KIE of on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
Related Links	http://dx.doi.org/10.1073/pnas.1209446110
Ending Page	2093
Page Count	6
Starting Page	2088
File Format	PDF
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	6
Volume Number	110
Language	English
Publisher	National Academy of Sciences
Publisher Date	2013-02-05
Access Restriction	Open
Rights Holder	National Academy of Sciences
Subject Keyword	General Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII.

Mechanistic Studies of the Radical SAM Enzyme DesII: EPR Characterization of a Radical Intermediate Generated During Its Catalyzed Dehydrogenation of TDP-D-Quinovose

Stoichiometry of the Redox Neutral Deamination and Oxidative Dehydrogenation Reactions Catalyzed by the Radical SAM Enzyme DesII

Mechanistic Studies of the Radical SAM Enzyme DesII with TDP-d-Fucose**

Mechanistic Investigation of the Radical S-Adenosyl-l-methionine (SAM) Enzyme DesII Using Fluorinated Analogs

EPR Characterization of the Radical SAM Enzyme HydG, Actinides, and Organic Radicals

Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

A Consensus Mechanism for Radical SAM-Dependent Dehydrogenation? BtrN Contains Two [4Fe–4S] Clusters †

Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

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EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII.

Mechanistic Studies of the Radical SAM Enzyme DesII: EPR Characterization of a Radical Intermediate Generated During Its Catalyzed Dehydrogenation of TDP-D-Quinovose

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EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII