Abstract
The glyoxalase pathway intermediate S-d-lactoylglutathione was recently implicated in protein post-translational modifications in animal systems. Here, we examined the spontaneous modification of the Arabidopsis thaliana cytosolic glyceraldehyde-3-phosphate dehydrogenase C1 (GAPC1) by this compound. Incubation of GAPC1 with S-d-lactoylglutathione resulted in the inhibition of enzyme activity. The inhibitory effect was concentration dependent and increased at alkaline pHs. Furthermore, the inhibition of GAPC1 by S-d-lactoylglutathione was favored by oxidative conditions and reversed by reduction with dithiothreitol. Analyses of the S-d-lactoylglutathione-treated protein by nanoLC-MS/MS revealed S-glutathionylation of its two Cys residues and N-lactoylation of six Lys residues. Protein structure predictions showed that the double S-glutathionylation is accommodated by the GAPC1 catalytic pocket, which likely explains enzyme inhibition. N-lactoylated sites overlap partially with previously reported N-acetylated sites at the surface of the GAPC1 tetramer. The efficiency of cytosolic glutaredoxin and thioredoxin isoforms was tested for reversing the S-d-lactoylglutathione-induced modification. In these assays, recovery of GAPC1 activity after inhibition by S-d-lactoylglutathione treatment was used as indicator of efficiency. The results show that both types of redoxins were able to reverse inhibition. We propose a model describing the mechanisms involved in the two types of post-translational modifications found on GAPC1 following exposure to S-d-lactoylglutathione. The possible involvement of these findings for the control over glycolytic metabolism is discussed.