Proton Transfer via Arginine with Suppressed pKa Mediates Catalysis by Gentisate and Salicylate Dioxygenase

Abstract

Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) facilitate aerobic degradation of aromatic rings by inserting both atoms of dioxygen into their substrates, thereby participating in global carbon cycling. The role of acid–base catalysts in the reaction cycles of these enzymes is debatable. We present evidence of the participation of a proton shuffler during catalysis by GDO and SDO. The pH dependence of Michaelis–Menten parameters demonstrates that a single proton transfer is mandatory for the catalysis. Measurements at variable temperatures and pHs were used to determine the standard enthalpy of ionization (ΔH_ion°) of 51 kJ/mol for the proton transfer event. Although the observed apparent pK_a in the range of 6.0–7.0 for substrates of both enzymes is highly suggestive of a histidine residue, ΔH_ion° establishes an arginine residue as the likely proton source, providing phylogenetic relevance for this strictly conserved residue in the GDO family. We propose that the atypical 3-histidine ferrous binding scaffold of GDOs contributes to the suppression of arginine pK_a and provides support for this argument by employing a 2-histidine-1-carboxylate variant of the enzyme that exhibits elevated pK_a. A reaction mechanism considering the role of the proton source in stabilizing key reaction intermediates is proposed.