Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O-methyltransferase reaction

Abstract

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 15-20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can 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.

Keywords: enzyme mechanism; hydrogen deuterium exchange; methyl transfer; protein flexibility.

Fig. 1.

Examples of different patterns of HDX responses to temperature (types I, II-A, II-B). Time courses of percent deuterium incorporation vs. time at 10 °C (black, triangle), 20 °C (yellow, square), and 37 °C (red, dots). The text includes details of these assignments, and Dataset S1 shows plots of all observed peptides.

Fig. 2.

The mapping of HDX patterns to the crystal structure of WT COMT. Shown is the structure of a ternary complex of COMT with AdoMet cofactor and dinitrocatechol substrate (PDB ID code 3BWM) (36) with the following color scheme for HDX in WT enzyme: type I, cyan; type II-A, yellow; type II-B, raspberry. Mg²⁺ metal is in orange. Because the reported HDX experiments represent the apo protein, we have shown the AdoMet and substrate as thin lines rather than the usual stick representation (see Fig. 4). Data for the region containing the site of mutation (gray) were not complete, but, based on two temperatures, appeared to correspond to type I behavior (see text).

Scheme 1.

Kinetic pathway for COMT, illustrating the ordered binding of AdoMet, Mg²⁺, and substrate, followed by the ordered release of AdoHcy, Mg²⁺, and product.

Fig. 3.

Representative type II-B peptides showing variations in HDX between WT and Y68A COMT. Time courses of percent deuteration vs. time are shown for 37 °C (red), 20 °C (yellow), and 10 °C (black). In all cases, deuteration time courses were comparable between Y68A (Right) and WT (Left) at 37 °C and 10 °C, but markedly reduced deuteration was seen in Y68A relative to WT at 20 °C, revealing a break in the temperature dependence of HDX uptake introduced by the mutation.

Fig. 4.

The mapping of variations in HDX behavior between WT and Y68A. The regions colored magenta correspond to positions showing a break in the temperature dependence of HDX in Y68A at 20 °C. Color coding for the remainder of the protein is as in Fig. 2.

Fig. 5.

Temperature dependences for kinetic parameters of WT and Y68A COMT. Shown are Arrhenius plots of lnk_cat vs. 1/T for (A) WT and (B) Y68A COMT and lnk_cat/K_m vs. 1/T for (C) WT and (D) Y68A. Whereas WT shows a single straight line at all temperatures, Y68A reveals a breakpoint in slope at ca. 15 to 20 °C. Data are summarized in SI Appendix, Tables S3 and S4, and tests of fitting are shown in SI Appendix, Fig. S3.

Fig. 6.

Dissociation constants (K_d) for AdoMet (reactant) and AdoHcy (product) with COMT at varying temperatures. Plots of K_d vs. temperature are shown for WT and Y68A enzymes as indicated. Data are summarized in SI Appendix, Tables S5 and S6; the data points in brackets for WT COMT are considered outliers, as described in SI Appendix, Table S6.

Fig. 7.

A model for the COMT reaction integrating the HDX and kinetic findings. (A) The active site of COMT, illustrating the proposed duality of roles for Tyr68 in controlling the methyl donor–acceptor distance along the reaction coordinate (dashed black line) and the sampling of multiple conformational substates involving side chains orthogonal to the reaction coordinate (dashed light blue line, Inset). Structure is based on PDB ID code 3BWM (36), not showing dinitro groups on the catechol. (B) Space filling model of COMT showing the dynamical regions of the protein that extend in orthogonal directions from the protein solvent interface toward the reacting atoms in the active site.