Enzymatic methyl transfer: role of an active site residue in generating active site compaction that correlates with catalytic efficiency

Abstract

Human catechol-O-methyltransferase (COMT) catalyzes a methyl transfer from S-adenosylmethionine (AdoMet) to dopamine. Site-specific mutants at three positions (Tyr68, Trp38, and Val108) have been characterized with regard to product distribution, catalytic efficiency, and secondary kinetic isotope effects. The series of mutations at Tyr68 within wild-type protein and the common polymorphic variant (Val108Met) yields a linear correlation between the catalytic efficiency and the size of the secondary kinetic isotope effect. We conclude that active site compaction in COMT is modulated by a proximal side chain residing behind the sulfur-bearing methyl group of AdoMet. These findings are discussed in the context of the active site compression that has been postulated to accompany enzyme-supported hydrogen tunneling.

Figure 1

Active site of human WT s-COMT complexed with catechol (magenta), AdoMet (blue), Mg²⁺ (yellow), residues Glu6 (orange), Trp38 (cyan), Tyr68 (green) and Val108 (red) shown in stick representation (PDB: 3BWM). Distance values (in firebrick) are underlined with unit Å.

Figure 2

Relationship between the k_cat/K_m and 2° KIE for 3-O-methylation catalyzed by Y68 mutants of s-COMT; data are presented for both WT and the V108M variants. (a) k_cat/K_m(Dopamine) vs. the corresponding 2° KIE (k_CH3/k_CT3); r² = 0.96. (b) k_cat/K_m (AdoMet) vs. the corresponding 2° KIE (k_CH3/k_CT3); r₂ = 0.90. Data in black refer to the WT series; data in red refer to the V108M series.

Scheme 1