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

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 GadBH465A variant to avoid the masking effect of His465 at pH > 5. 5. In addition, GadBD86N-H465A was shown to be less sensitive than GadBH465A 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 GadBD86N-H465A interesting also for effective biobased synthesis of GABA.