Biochemical properties and redox sensitivity of mutants involved in the proton relay of GAPc from Arabidopsis thaliana

GAPc is a subset of enzymes of the family glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a glycolytic enzyme conserved in all living organisms. In plants, it also functions as a redox regulator, by switching on cellular stress response against oxidant molecules. Its active site contains a strictly conserved cysteine (Cys149), which directly participates in the catalysis, but is also prone to oxidative modifications. Cysteine oxidation is enhanced by the proton relay network that surrounds the active site of GAPc. This coordinated network permits the passage of protons between neighboring amino acids and rapidly stabilises the enzyme in the presence of oxidant molecules, such as H2O2. Two of these amino acids are threonine 157 (Thr157), that creates hydrogen bonds, and tyrosine 314 (Tyr314), which interacts with catalytic cysteine. In this work, we investigated the role of two of the amino acids surrounding the active site by site-directed mutagenesis. Residues Thr157 and Tyr314 were substituted with an alanine, and mutagenized plasmids were used to transform E. coli for recombinant overexpression. Purification of mutants Thr157A and Tyr314A was performed by metal-affinity chromatography. Subsequently, their specific activity and the sensitivity to hydrogen peroxide (H2O2) have been analyzed. Finally, their structural stability has been evaluated through turbidity assays, without exposure to oxidative stress. We discovered that, when Tyr314 is mutated, the protein has a tendency of aggregation or misfolding, which leads to a decreased activity of the enzyme. On the other hand, the mutant for Thr157 behaves similarly to the wild-type, but shows a slower catalytic rate and a reduced sensitivity to H2O2-dependent oxidation. Taken together, these findings highlight the role of proton relay in oxidative regulation: as the amino acids surrounding the active site are disrupted by removing the group needed for proton transfer, the oxidation of Cys149 is much slower likely because the effective coordination between residues is missing.