Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis

Abstract

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.

Keywords: feedback loop; glycolysis; phosphoenolpyruvate; pyruvate kinase; triosephosphate isomerase.

Figure 1.

Co-crystal structure of TPI with bound PEP. (a) Schematic of the TPI–PEP crystallographic structure. PEP locates in the active centre of both subunits in the asymmetric TPI dimer. (b) The catalytic pocket of TPI bound to PEP. Catalytic residues are highlighted in yellow, PEP in red, isoleucine 170 in green. (c) Stereoscopic illustration of the PEP binding site environment including a difference map in which PEP has been removed from the model and was refined against the experimental data for five cycles. The map has been contoured at 4 s.d. and reveals positive density for the missing ligand.

Figure 2.

The TPI inhibitor PEP and the TPI substrate DHAP have similar interaction sites. (a) Contact distances between TPI and its substrate DHAP, and (b) the interactions of TPI and PEP in the active site. PEP and DHAP are in contact with similar principal residues. Distances are given in Å. Green balls, phosphate; grey balls, carbon; red balls, oxygen. The red circles indicate residues in close proximity to the ligand. Illustrations were prepared using LigPlot. (c) PEP and DHAP bind similarly to the TPI active site. Rabbit TPI bound to PEP, overlaid with the location of the TPI substrate DHAP as determined by Jogl et al. [28] as surface representation. Yellow areas highlight catalytically active residues; PEP: red; DHAP: blue.

Figure 3.

PEP competes with G3P for binding to human TPI. (a) Thermal stability of human TPI and active-site mutants TPI_Ile170Val and TPI_Ile170Thr in the presence of increasing PEP concentrations. PEP stabilized the three-enzyme species indicative for binding; TPI_Ile170Val and TPI_Ile170Thr were stabilized to an increased extent. (b) Thermal stability of human TPI mutants to increasing G3P concentrations; increased thermal stability of TPI_Ile170Thr indicated augmented affinity for G3P. (c) PEP dose–response curve in the presence of G3P. PEP binding was competitive against G3P in human TPI and TPI_Ile170Thr, but did not influence the thermal stability of TPI_Ile170Val.

Figure 4.

PEP inhibits the catalytic activity of TPI. (a) TPI_Ile170Val and TPI_Ile170Thr have reduced catalytic activity, TPI_Lys13Arg is inactive. Enzyme activity expressed as substrate conversion rate in micromoles per minute and microgram protein. (b) Enzymatic properties of TPI, TPI_Ile170Val, TPI_Ile170Thr and their inhibition by PEP. (c) Substrate titration curves of G3P (black curves, to be read from left to right) on TPI and its mutant enzymes, as well as inhibitor titration curves for PEP (blue curves, to be read from right to left). Substrate/inhibitor saturation was used to calculate V_max, K_m (G3P titrations), and IC₅₀ and K_i values (PEP titrations) (inset table).

Figure 5.

Human TPI_Ile170Val and TPI_Ile170Thr complement for yeast TPI and are catalytically active. (a) TPI, TPI_Ile170Val and TPI_Ile170Thr, but not TPI_Lys13Arg, complement for yeast TPI1. In a plasmid shuffle experiment, Δtpi1 cells carrying a counterselectable TPI-encoding plasmid were transformed with a centromeric plasmid (minichromosome) encoding the indicated TPI mutants. Transformed cells were then transferred to 5'FOA to induce loss of the counterselectable plasmid. Only cells containing a functional TPI copy on the minichromosome are viable on glucose media after counterselection. Human TPI, TPI_Ile170Val and TPI_Ile170Thr complemented for a loss of the TPI plasmid, but TPI_Lys13Arg did not. (b) TPI activity in yeast whole-cell extracts. Substrate conversion rates as normalized to total protein content. TPI_Ile170Val and TPI_Ile170Thr have lower activity than wild-type TPI. (c) Increased expression levels of TPI_Ile170Val and TPI_Ile170Thr in yeast as revealed by immunoblotting of whole-cell extracts using a TPI-specific antibody [35]. The amount loaded onto the SDS-PAGE gel was normalized to total protein, comparable loading was evaluated by Ponceau Red staining of the blotting membrane.

Figure 6.

Low TPI activity increases PPP metabolite load and causes oxidant resistance and heat sensitivity. (a) Concentrations of glycolytic and PPP metabolites in the human TPI_Ile170Val and TPI_Ile170Thr mutants relative to yeast expressing human wild-type TPI. PPP and glycolytic metabolites were quantified by LC-MS/MS. PPP metabolites are increased in the TPI mutants. Absolute values are given in the electronic supplementary material, figure S4. (b) TPI_Ile170Val and TPI_Ile170Thr mediate increased tolerance to oxidizing agents. Overnight cultures of the indicated yeast strains were diluted to an OD₆₀₀ = 3 and spotted onto SC^−His agar plates containing the oxidants. Glucose 6-phosphate dehydrogenase (Zwf1) encodes the enzyme for the first step in the non-reversible oxidative PPP shunt and produces NADPH. Its deletion abolishes the oxidant resistance phenotype of cells expressing TPI_Ile170Val or TPI_Ile170Thr. Sol3 and Sol4 catalyse the second step of the PPP and their deletion reduced oxidant resistance on H₂O₂; a protective effect of TPI_Ile170Val was detected in Δsol3 yeast while causing H₂O₂ sensitivity in Δsol4 yeast. (c) TPI mutants are heat-sensitive. Overnight cultures were diluted to an OD₆₀₀ = 0.2 and exposed, or not exposed, to 50°C for 5 min and growth was monitored for 25 h after heat exposure. The duration until growth was re-established (lag phase) was used as an inverse indicator for heat resistance. The lag phase was prolonged in the 50°C exposed TPI mutants compared with isogenic yeast cells expressing wild-type TPI.