Enzymatically-regulated protein post-translational modification and allosteric protein regulation via non-covalent binding interactions represent conserved regulatory mechanisms in biology. At the confluence of these two central mechanisms, we hypothesized that endogenous, reactive primary metabolites might regulate protein function via non-enzymatic covalent modification of proteins. One such metabolite, the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG), contains an acyl-phosphate functional group, which has been previously shown to react with primary amines. As such, we predicted that this central metabolite would form stable 3-phosphoglyceryl amide adducts with reactive lysine side-chains in the proteome. In vitro conversion of 3-phosphoglyceraldehyde to 1,3-BPG with the glycolytic enzyme GAPDH led to the formation of 3-phosphoglyceryl-lysine (pgK) adducts on GAPDH itself, which was confirmed by isoelectric focusing and high-resolution LC-MS/MS experiments. Subsequent proteomic profiling of human cell lines has identified more than 80 endogenous pgK-modification sites, which include a strong enrichment of glycolytic enzymes among other protein classes. Isotopic labeling experiments have confirmed that the pgK modification originates from glucose and that pgK-protein levels in the cell are dynamically regulated by altered glycolytic flux. Inspection of published structures revealed that several endogenous pgK sites are found in or around the active site of glycolytic enzymes, raising the possibility of a regulatory role. In line with this, we have demonstrated that pgK modification or mutation of labeled lysines in GAPDH and ENO1 results in significantly reduced enzymatic activity. Collectively, these data have identified a novel, reactivity-driven, post-translational lysine modification by an endogenous metabolite and suggests that this might serve as a new regulatory feedback/forward mechanism in glycolysis, where pgK-levels both respond to and regulate glycolytic flux.

Post-translational modification of lysines by an endogenous, reactive glycolytic metabolite dynamically regulates glycolytic enzymes

243rd National Meeting (2012)

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