Anton Simeonov Ph.D. National Institute of Health

In this literature review, researchers discuss a PTM they believe was formed through an enzyme-independent direct chemical reaction.

The number of reported posttranslational modifications (PTMs) has grown dramatically during the past decade. Almost universally, however, these covalent modifications are installed onto protein amino acid side chains through the direct catalytic action of specific enzymes. Here, the Cravatt team* reports on a new PTM that appears to be formed through an enzyme-independent direct chemical reaction.

The authors studied the propensity of reactive metabolic intermediates to form covalent adducts with proteins and focused their investigation on 1,3-bisphosphoglycerate (1,3-BPG), a product of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed conversion of 3-phosphoglyceraldehyde. Reaction of 1,3-BPG with lysine residues on proteins was expected to produce 3-phosphoglyceryl-lysine (pgK) modifications (Figure 1).

Figure 1. 1,3-BPG forms a stable, covalent modification on lysines of GAPDH in vitro. (A) pgK formed by reaction of a lysine e-amine with the acylphosphate functionality in 1,3-BPG. (B) Spectral counts of pgK-modified tryptic peptides detected by LC-MS/MS analyses of GN, GG, and GGN GAPDH enzymatic reactions (average of two independent experiments). (C) MS/MS spectra of the in vitro GGN-GAPDH–derived (left) and synthetic (right) doubly charged tryptic peptide VV(pg)KQASEGPLK. Observed b-, y-, and relevant parent ions, as well as products of dehydration (°) or ammonia loss (*) are labeled. Asterisk (*) within peptide sequences denotes the pgK-modified lysine. (D) The most frequently detected pgK-modification sites (K107, K194, and K215) surround the active site of GAPDH (PDB accession no. 1ZNQ). (E) Western immunoblot (IB) with antibody against GAPDH in GG and GGN-GAPDH reactions after IEF analysis. Data are from a representative experiment of three independent experiments. 1,3-BPG, 1,3-bisphosphoglycerate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pgK, 3-phosphoglyceryl-lysine; LC, liquid chromatography; MS/MS, tandem mass spectrometry; GN, substrate; GG, cofactor; GGN, substrate and cofactor; PDB, Protein Data Bank; IEF, isoelectric focusing.

Figure 1

After performing the GAPDH reaction using purified enzyme, the authors analyzed the enzyme itself for potential pgK modifications received during the multiple turnovers and indeed three lysines (residues 107, 194, and 215) were identified as the sites of most frequent modifications. Detailed analyses using human cell lines and mouse tissues revealed pgK modifications in multiple enzymes primarily involved in metabolism and associated processes such as glycolysis, amino acid metabolism, and oxidative phosphorylation (Figure 2).

Figure 2. Functional distribution of pgK modification sites in human cells and mouse tissues. (A, B) Modification site, peptide sequence, and associated annotation for representative endogenous pgK-modified proteins from human cell lines (A) and mouse liver (B). “(pg)K” denotes the pg-modified lysine. (C, D) Gene ontology biological process categories (GOTERM_BP) and KEGG pathways enriched among pgK-modified proteins in human cell lines (C) and mouse liver (D) by DAVID bioinformatic analysis. (E) Schematic of observed pgK-modified enzymes in glycolysis. Glycolytic enzymes containing at least one pgK site are shown in red, others are shown in gray.

Figure 2

Because the installation of phosphoglyceride group onto lysines increases the size and flips the side-chain charge, the authors surmised that the pgK PTM has the potential to affect the structure and function of the modified protein. Indeed, the team further showed that the pgK status of proteins was coupled to glucose metabolism and that the pgK modification was reversible. The important discovery of this self-installing PTM will likely spur follow-up in vivo studies of the potential role of pgK modification in a range of metabolic diseases and cancer.

*Abstract from Science 2013, Vol. 341: 549–553

The posttranslational modification of proteins and their regulation by metabolites represent conserved mechanisms in biology. At the confluence of these two processes, we report that the primary glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) reacts with select lysine residues in proteins to form 3-phosphoglyceryl-lysine (pgK). This reaction, which does not require enzyme catalysis, but rather exploits the electrophilicity of 1,3-BPG, was found by proteomic profiling to be enriched on diverse classes of proteins and prominently in or around the active sites of glycolytic enzymes. pgK modifications inhibit glycolytic enzymes and, in cells exposed to high glucose, accumulate on these enzymes to create a potential feedback mechanism that contributes to the buildup and redirection of glycolytic intermediates to alternate biosynthetic pathways.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Science titled “Functional lysine modification by an intrinsically reactive primary glycolytic metabolite.” Authors of the paper are Moellering RE and Cravatt BF.

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