An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor

Abstract

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer–monomer dissociation Kd is estimated to be ∼0.9 µM with a slow dissociation rate (t1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such ‘allostatic’ regulation may be important in metabolic reprogramming and influencing cell fate.

Cancer‐associated mutations in human pyruvate kinase M2 impair enzyme activity

“…This HPLC separation method allows injection of enzyme at a relatively high concentration of 0.5 mgÁmL À1 , which is approximately 500-fold more concentrated than enzyme concentrations used for the kinetic assays. This provides an equivalent concentration of monomers (~86 lM) that is almost two orders of magnitude greater than the recently reported tetramer-dimer and tetramer-monomer dissociation constants of around 1 lM for wild-type PKM2 [18,39]. The nondilutive HPLC assay conditions should thus favor PKM2 tetramer formation and provide a more direct measure of tetramer stability than the functional AC 50 determinations.…”

“…HPLC size-exclusion chromatography evaluates relatively concentrated samples (0.5 mgÁmL À1 ) that are a better approximation of cellular conditions than is the kinetic assay, which requires significant dilution of this highly active enzyme (to approximately 1 lgÁmL À1 or 17 nM) to allow determination of initial rates. The in vivo concentration of pyruvate kinase has been reported as 172 µM or 10 mgÁmL À1 [56], a value well above the reported K D of 1 lM for tetramer-monomer and tetramer-dimer equilibria [18,39]. The HPLC sizeexclusion results were thus obtained from samples at concentrations above the K D for tetramer dissociation, while the kinetic assays can only be conducted with samples at concentrations below the K D which favors tetramer dissociation.…”

“…PKM2 activity is modulated by a host of regulatory inputs. Like PKL and PKR, FBP is a major allosteric activator of PKM2 [14,15], and its activity is also affected by other allosteric effectors, including thyroid hormone T 3 , serine, phenylalanine, and select other amino acids [7, [16][17][18][19]. FBP binding increases affinity of PKM2 for one of its substrates, PEP, and stabilizes the PKM2 tetramer in a fully active conformation [15,20].…”

Citrullination of pyruvate kinase by PADI1 and PADI3 regulates glycolysis and cancer cell proliferation

“…As described in the introduction, PKM2 isoform activity is positively regulated by serine (Ser), and FBP and negatively by Trp, Ala and Phe, thus coupling glycolytic flux to the level of critical intermediate metabolites (4)(5)(6). PKM2 allosteric regulation involves three distinct enzyme conformations [ (8,9,31) To test the above hypotheses, we asked if citrullination modulated glycolysis under different conditions. When cells were grown in absence of Ser, basal glycolysis was reduced and was no longer stimulated upon siCHD4 or PADI1/3 expression ( Fig.…”

“…Unlike the PKM1 isoform that is constitutively active, PKM2 activity is positively regulated by serine (Ser), fructose 1,6-biphosphate (FBP) an intermediate of the glycolytic pathway and succinylaminoimidazole-carboxamide riboside (SAICAR), an intermediate in de novo purine nucleotide synthesis (4,6,7). High levels of these molecules stimulate PKM2, but when their levels are lowered through excessive glycolysis, PKM2 activity is inhibited by amino acids such as tryptophan (Trp), alanine (Ala) and phenylalanine (Phe) that compete with Ser to allosterically regulate PKM2 activity (8)(9)(10).…”