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.