# Role of Aspartate 86 in the Catalytic Mechanism of Escherichia coli Glutamate Decarboxylase

## TL;DR

This study identifies Aspartate 86 as a key player in the catalytic mechanism of Escherichia coli glutamate decarboxylase, impacting substrate binding and product release.

## Contribution

The novel finding is that Asp86 is a major contributor to the solvent isotope effect and pH-dependent activity in EcGadB.

## Key findings

- Asp86 plays a significant role in substrate binding and product release in EcGadB.
- GadB_D86N-H465A remains active at higher pH levels (7–8) compared to the wild-type enzyme.
- The enzyme variant GadB_D86N-H465A is promising for biobased GABA synthesis due to improved product release.

## Abstract

In bacteria, the
pyridoxal 5′-phosphate (PLP)-dependent
enzyme glutamate decarboxylase (Gad) protects the cells exposed to
an acidic environment by consuming one proton/catalytic cycle during
the conversion of l-glutamate to γ-aminobutyrate (GABA)
and CO2. The Escherichia coli enzyme (EcGadB) is the best-characterized bacterial
Gad; its activity is maximal at pH 4–5 and undetectable at
pH ≥ 6.0, at which the active site is closed by His465. The
imidazole ring of this His residue, highly conserved in bacterial
Gad, becomes deprotonated as the pH increases above 5.0 and carries
out a nucleophilic attack on the PLP-Lys276 Schiff base. However,
when His465 is mutated, EcGadB activity still displays
pH dependence, indicating that other residues also play a role. Herein,
through a combination of spectroscopic and kinetic analyses, including
solvent kinetic isotope effect (SKIE) and proton inventory studies,
Asp86, another residue highly conserved in bacterial Gad, was shown
to play an important role in substrate binding and product release
and, unexpectedly, to be a major player in the large SKIE observed
in EcGadB. This was demonstrated by incorporating
the D86N substitution into the GadB_H465A variant to avoid the masking
effect of His465 at pH > 5.5. In addition, GadB_D86N-H465A was
shown
to be less sensitive than GadB_H465A to the pH increase occurring
during the decarboxylation, being still active in the pH range 7–8,
where glutamate solubility increases. This finding, together with
the enzyme’s improved ability to release the product, makes
GadB_D86N-H465A interesting also for effective biobased synthesis
of GABA.