MassIVE MSV000085213

Partial Public

Hydrogen Deuterium Exchange Defines Catalytically Linked Regions of Protein Flexibility in the Catechol O-Methyltransferase Reaction

Description

Human catechol-O-methyltransferase (COMT) has emerged as a model for understanding enzyme catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small molecule catecholate acceptors. Mutation of a single residue (Tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor-acceptor compaction within the activated ground state of the wild type enzyme (J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U. S. A. 112, 7954-7959, 2015). This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties, that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15-20C indicates changes in flexibility across approximately 40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20C, Tyr68A behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Acan be understood in the context of a mutationally-induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate. [doi:10.25345/C53X48] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: hydrogen deuterium exchange ; protein flexibility ; enzyme catalysis ; COMT ; HXMS ; HDX-MS

Contact

Principal Investigators: Natalie Ahn, University of Colorado Boulder, USA
Judith Klinman, University of California Berkeley, USA
Submitting User: jbalsbaugh
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