Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

Abstract

Allosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands. activity promotes pro-tumorigenic functions, including divergence of glucose carbons into biosynthetic and redox-regulating pathways that support proliferation and defence against oxidative stress 13,14,20,21 . Small-molecule activators, which render PKM2 constitutively active by overcoming endogenous inhibitory cues, attenuate tumour growth, suggesting that regulation of PKM2 activity, rather than increased PKM2 expression per se, is 4 important 13,20,22,23 . Therefore, PKM2 has emerged as a prototypical metabolic enzyme target for allosteric modulators and this has contributed to the renewed impetus to develop allosteric drugs for metabolic enzymes, which are expected to specifically interfere with cancer cell metabolism while sparing normal tissues 11 .The structure of the PKM2 protomer comprises N-terminal, A, B and C domains. Various ligands that regulate PKM2 activity bind to sites distal from the catalytic core, nestled between the A and B domains. The upstream glycolytic intermediate fructose-1,6-bisphosphate (FBP) binds to a pocket in the C-domain 24 and increases the enzymatic activity of PKM2, thereby establishing a feed-forward loop that prepares lower glycolysis for increased levels of incoming glucose carbons. Activation of PKM2 by FBP is associated with a decreased K M for the substrate PEP, while the k cat remains unchanged 25-28 , although some reports also find an elevated k cat 29,30 and the reason for this discrepancy remains unknown. Additionally, several amino acids regulate PKM2 activity by binding to a pocket in the TIM-barrel core 25,31,32 . Lserine (Ser) and L-histidine (His) increase, whereas L-phenylalanine (Phe), L-alanine (Ala), Ltryptophan (Trp), L-valine (Val) and L-proline (Pro) decrease the apparent affinity for PEP.Similar to FBP, the reported effects on k cat vary 25,29,31,33 . Furthermore, succinyl-5aminoimidazole-4-carboxamide-1-ribose 5′-phosphate (SAICAR) 34 ...

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

“…An activity‐based determination of FBP activation of refolded PKM2 protein yielded an AC 50 value of ~ 2 μ m (Fig. D), which is also comparable to previously reported values for PKM2 activation and binding of FBP to nonphosphorylated PKL , although somewhat higher than a more recently reported AC 50 of 118 n m . Despite the kinetic behavior of the refolded PKM2 in response to FBP being more consistent with past studies, Prep 2 was laborious and we sought an alternative preparation of native, bacterially expressed PKM2 that is largely free of bacterial FBP.…”

“…A). These results suggested that most of the enzyme was already bound to FBP from the bacteria despite purification as previously reported . Independent preparations of PKM2 using this approach occasionally produced enzyme that showed some FBP activation; however, this variability in FBP activation complicated detailed kinetic analysis.…”

“…The high affinity of PKM2 for FBP is shared by other mammalian pyruvate kinase isoforms such as PKL, but not by the pyruvate kinase isoforms of unicellular organisms. The values reported elsewhere for half‐maximal binding or half‐maximal activation of mammalian pyruvate kinases are in the sub‐ to low‐micromolar range (0.34–7.5 μ m ) , with the most recent work reporting K D = 25.5 n m for direct binding of FBP to PKM2 (after correction for copurified FBP) and AC 50 = 118 n m for FBP activation of wild‐type PKM2 . While the FBP AC 50 value reported in the present study for WT PKM2 (48.7 n m , Fig.…”

“…A reported value for the intracellular FBP concentration in mammalian cells is 80 μ m , which is at least one order of magnitude greater than the concentration required for half‐maximal activation of PKM2. Thus, in the absence of other inputs, FBP binding to mammalian pyruvate kinases may be saturated in many cellular conditions in vivo . In contrast, the K 0.5 values for FBP activation are around 45 μ m for S. cerevisiae pyruvate kinase and 70 μ m for E. coli pyruvate kinase , making the yeast and E. coli pyruvate kinase isoforms better suited to respond to changes in intracellular FBP occurring in the physiological concentration range.…”

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