Functional Lysine Modification by an Intrinsically Reactive Primary Glycolytic Metabolite

Enzyme regulation is an important process in controlling metabolic flux.  The two ways in which this is thought to happen is by inhibiting an enzyme with a small molecule or through a covalently-bound post-translational modification.  This second process is thought to require an enzyme to form this covalent bond.  Moellering and Cravatt challenge this paradigm by showing that inherently reactive intermediates can form covalent bond modificatins without the use of an enzyme.  They do this using 1,3-bisphosphoglycerate (1,3-BPG), which is formed by the enzyme GAPDH in the process of glycolysis.  This intermediate was thought to be a prime candidate for study because its acylphosphate group makes it inherently reactive, especially with lysine residues.  If 1,3-BPG reacts with lysine, it would form 3-phosphoglyceryl-lysine (pgK).

(A) Intracellular glucose and bisphosphoglycerate (BPG, aggregate of both 1,3- and 2,3-isomers) levels from cells grown at indicated glucose concentrations for 24 hours. (B) Antibody against pgK (α-pgK) IB and Coomassie-stained gel of proteomes from HEK293T cells grown at indicated glucose concentrations. (C and D) α-pgK IB of α-FLAG–enriched ENO1 (C) and GAPDH (D) expressed in HEK293T cells grown at indicated glucose concentrations. Shown below each blot is a graph of the average relative α-pgK band intensities (n = 4 per group). (E) Representative SILAC chromatograms, MS1 isotope envelopes, and corrected (corr.) area ratios for non-pgK (left) and pgK-modified (right) peptides from ENO1. Integration area is shown within green bars; asterisk (*) in the chromatogram signifies the triggered MS2 scan. (F) Average SILAC ratios for pgK343-containing and non-pgK ENO1 peptides from cells grown at indicated glucose concentrations. Horizontal line and whiskers represent the mean and 10 to 90% confidence intervals, respectively. (G) IB of IEF-focused ENO1 from MCF7 cells grown at the indicated glucose concentrations. Plot shows the IEF-focused ENO1 pI distributions quantified by densitometry. (H) Spectral count values for pgK-modified enolase peptides across nine mouse tissues (table S3). Data represent means ± SEM; statistical significance was determined by two-way t tests with a Bonferroni correction for (C) and (D) and Welch’s correction for (F): *P < 0.05; **P < 0.01; ***P < 0.005.  Science. 2013. 341(6145):549-553
(A) Intracellular glucose and bisphosphoglycerate (BPG, aggregate of both 1,3- and 2,3-isomers) levels from cells grown at indicated glucose concentrations for 24 hours. (B) Antibody against pgK (α-pgK) IB and Coomassie-stained gel of proteomes from HEK293T cells grown at indicated glucose concentrations. (C and D) α-pgK IB of α-FLAG–enriched ENO1 (C) and GAPDH (D) expressed in HEK293T cells grown at indicated glucose concentrations. Shown below each blot is a graph of the average relative α-pgK band intensities (n = 4 per group). (E) Representative SILAC chromatograms, MS1 isotope envelopes, and corrected (corr.) area ratios for non-pgK (left) and pgK-modified (right) peptides from ENO1. Integration area is shown within green bars; asterisk (*) in the chromatogram signifies the triggered MS2 scan. (F) Average SILAC ratios for pgK343-containing and non-pgK ENO1 peptides from cells grown at indicated glucose concentrations. Horizontal line and whiskers represent the mean and 10 to 90% confidence intervals, respectively. (G) IB of IEF-focused ENO1 from MCF7 cells grown at the indicated glucose concentrations. Plot shows the IEF-focused ENO1 pI distributions quantified by densitometry. (H) Spectral count values for pgK-modified enolase peptides across nine mouse tissues (table S3). Data represent means ± SEM; statistical significance was determined by two-way t tests with a Bonferroni correction for (C) and (D) and Welch’s correction for (F): *P < 0.05; **P < 0.01; ***P < 0.005.
Science. 2013. 341(6145):549-553

The authors began by combining GAPDH with its substrate and cofactor to produce 1,3-BPG.  GAPDH was then separated using LC/MS, and they found that GAPDH was, indeed, modified to form pgK residues.  To determine the scope of 1,3-BPG modification, the authors incubated cells with a high concentration of glucose.  They found that there were a greater number of pgK residues in several glycolytic enzymes, including enolase 1, relative to control cells.

Now that the authors determined that pgK residues can form without the use of an enzyme and that it can be found on multiple enzymes, they sought to show that these pgK-modified proteins will be inactivated. They performed an in vitro study where 1,3-BPG was removed by dialysis.  This showed a lower Km than when 1,3-BPG remained in solution.  Therefore, Moellering and Cravatt demonstrate that when 1,3-BPG is at high levels, it will turn off enzymes by forming covalent bonds without the aid of another enzyme.  This offers a new method by which enzymes can be modified and, therefore, regulated.

http://www.sciencemag.org/content/341/6145/549.full

One Reply to “Functional Lysine Modification by an Intrinsically Reactive Primary Glycolytic Metabolite”

  1. Cool story, Hansel.

    GAPDH is so useful and interesting. Thank you for opening my eyes to ways of GAPDH.

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