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

## TL;DR

This study identifies arginine as the proton source in the catalytic mechanism of GDO and SDO enzymes, which break down aromatic compounds.

## Contribution

The paper reveals that arginine, not histidine, is the proton shuffler in GDO and SDO, supported by thermodynamic and phylogenetic evidence.

## Key findings

- A single proton transfer is essential for catalysis, with a ΔHion° of 51 kJ/mol.
- Arginine, not histidine, is the likely proton source due to suppressed pKa in the GDO family.
- A 2-histidine-1-carboxylate variant shows elevated pKa, supporting the role of the GDO scaffold in suppressing arginine pKa.

## 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 (ΔHion°) of 51
kJ/mol for the proton transfer event. Although the observed apparent
pKa in the range of 6.0–7.0 for
substrates of both enzymes is highly suggestive of a histidine residue,
ΔHion° 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 pKa and
provides support for this argument by employing a 2-histidine-1-carboxylate
variant of the enzyme that exhibits elevated pKa. A reaction mechanism considering the role of the proton
source in stabilizing key reaction intermediates is proposed.