Mediation of donor–acceptor distance in an enzymatic methyl transfer reaction

Abstract

Enzymatic methyl transfer, catalyzed by catechol-O-methyltransferase (COMT), is investigated using binding isotope effects (BIEs), time-resolved fluorescence lifetimes, Stokes shifts, and extended graphics processing unit (GPU)-based quantum mechanics/molecular mechanics (QM/MM) approaches. The WT enzyme is compared with mutants at Tyr68, a conserved residue that is located behind the reactive sulfur of cofactor. Small (>1) BIEs are observed for an S-adenosylmethionine (AdoMet)-binary and abortive ternary complex containing 8-hydroxyquinoline, and contrast with previously reported inverse (<1) kinetic isotope effects (KIEs). Extended GPU-based computational studies of a ternary complex containing catecholate show a clear trend in ground state structures, from noncanonical bond lengths for WT toward solution values with mutants. Structural and dynamical differences that are sensitive to Tyr68 have also been detected using time-resolved Stokes shift measurements and molecular dynamics. These experimental and computational results are discussed in the context of active site compaction that requires an ionization of substrate within the enzyme ternary complex.ethyltransferases are widely distributed in nature, playing critical roles in metabolic transformations, natural product biosynthesis (1, 2), and cellular regulation via the reversible methylation of proteins and nucleic acids (3-5). The ability to understand the origin of catalysis within this class of reactions is crucial to any efforts at protein redesign (6, 7) or inhibition (5,8,9). Catechol-O-methyltransferase (COMT), a drug target for a number of neurological diseases, emerged early as a prototype for mechanistic investigations (8,10,11). A compelling and unusual feature of COMT catalysis is the presence of a large inverse kinetic isotope effect (KIE) during transfer of the methyl group from the cofactor S-adenosylmethionine (AdoMet) to catechol acceptor. Comparison of the enzymatic behavior with a model reaction in solution led to the proposal of a role for transition state compression in enzymatic rate acceleration (12-18).Over the past decade, there has been a major shift in focus away from a historical interpretation of enzyme catalysis within the context of static 3D protein structures. Increasingly, protein motions are seen as integral to protein function at every level, from ligand binding to allosteric control and enzyme catalysis (19)(20)(21)(22). Models for catalysis that depend on such protein motions have been particularly important in the area of C-H activation, where the transfer of hydrogen by tunneling mechanisms implicates a critical dependence of the reaction rate on barrier width, and not just barrier height (23,24). Achievement of the tunneling-ready state requires a transient sampling of enzymatic ground states that achieves a reduced distance between the H-donor and acceptor (25). This feature raises the question of whether the reported inverse KIE in COMT could result from ground state interactions that bring about catalytically re...

Influence of Equatorial CH⋅⋅⋅O Interactions on Secondary Kinetic Isotope Effects for Methyl Transfer

“…This finding indicates that CH⋅⋅⋅O hydrogen bonding in AdoMet‐dependent methyltransferases as noted by Trievel and co‐workers1 may also modulate 2° KIEs, and that factors other than axial compression might contribute, at least partially, to the intriguing KIE variations for catechol‐ O ‐methyltransferase (COMT) and its mutant structures reported recently by Klinman and co‐workers 2, 3…”

“…However, hybrid QM/molecular‐mechanical (MM) computational simulations of this KIE for methyl transfer in solution and in the active site of COMT did not support the compression hypothesis: the trend in the KIEs was reproduced but without any significant difference in the average distance between the methyl donor and acceptor atoms in the corresponding TSs 6, 7. Nonetheless, an apparent trend in recent experimental T 3 KIEs for wild‐type and mutant COMTs has been interpreted as new evidence for compression 2, 3. Meanwhile, the functional importance of unconventional CH⋅⋅⋅O hydrogen bonding in AdoMet‐dependent methyltransferases has been noted from a survey of high‐resolution crystal structures,2 but the possible influence of these interactions on KIEs for methyl transfer is unknown.…”

“…It is known, however, that CD bond stretching frequencies are sensitive to the local electric field within a protein environment 16. Klinman and co‐workers have reported T 3 KIEs on k cat / K m in human COMT: 0.791±0.012 for the wild‐type enzyme and 0.822±0.021 and 0.850±0.012 for its Y68F and Y68A mutants, respectively 2, 3. The trend in these KIEs has been interpreted in terms of mediation of the distance between the methyl donor and acceptor groups,3 or “active‐site compaction”.…”

“…Klinman and co‐workers have reported T 3 KIEs on k cat / K m in human COMT: 0.791±0.012 for the wild‐type enzyme and 0.822±0.021 and 0.850±0.012 for its Y68F and Y68A mutants, respectively 2, 3. The trend in these KIEs has been interpreted in terms of mediation of the distance between the methyl donor and acceptor groups,3 or “active‐site compaction”. In terms of the cage model presented herein, the variations in KIE for COMT mutants would be attributed to changes in r ax , whereas we now suggest a significant role for changes in r eq which may lead to variations in KIE of similar magnitudes to those reported by Klinman and co‐workers.…”

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

“…At this point it is important to address a new twist in the argument about the NAC effect in COMT, where Klinman and coworkers 104 introduced a problematic analysis that is likely to confuse the uninitiated reader. More specifically, the authors of Ref.…”