Allosteric Mechanism of Pyruvate Kinase from Leishmania mexicana Uses a Rock and Lock Model

Abstract

Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 o rigid body rocking motion of the A-and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.Allosteric regulation ("the second secret of life" quotation ascribed to Jacob Monod) (see Ref. 1) controls many important cellular processes, including signal transduction, transcription, and metabolism (2). It describes the effect of binding one ligand on the subsequent binding of a second ligand at a topographically distinct site. Human pyruvate kinase (hPYK) 2 provides a striking example of the significance of allosteric regulation: a splicing switch of the primary RNA transcript to yield the M1 or M2 isoenzymes is now known to be responsible for the Warburg effect in cancer (3, 4) and opens up the possibility of developing isoform specific therapeutics (5). Additional mechanisms of activity regulation, including the binding of amino acids, phosphorylation, and the binding of oncoproteins, may provide further therapeutic approaches for targeting hPYK (6, 7).Most allosterically regulated proteins are enzymes in which the binding of an activator or inhibitor to the effector site can affect the binding of a substrate at the active site. The MonodWyman-Changeux model of allostery (8) suggests that oligomeric enzymes undergo symmetrical transitions (classically between the T-and R-states) 3 (36) that can be stabilized by ligand binding. However, there are now examples of allosteric control that do not show obvious conformational change (9), and a growing body of work exists to support the idea that flexible regions of molecules (which exist as an ensemble of conformers) may undergo allosteric regulation by changes in the conformer population (10). There are examples of allosteric enzymes in which the same protein has been captured in both a T-state (which has low affinity for substrate) and an R-state (which has higher affinity for substrate), and some structural insight has been obtained by the study of at least one of the allosteric states of Ͼ50 proteins (10 -12). However, a full understanding of the allosteric effect requires structural information on each of four states: (i) apoenzyme, (ii) active site complex, (iii) eff...

Pyruvate kinases have an intrinsic and conserved decarboxylase activity

“…Crystal soaking with oxalate reveals that the binding mode of oxalate at the active site of TbPYK is almost identical to the binding mode of PEP, pyruvate and OAA analogues, suggesting its action as a competitive inhibitor for both decarboxylase and kinase activities (Figures 5e, 5f and 6). Moreover, the binding mode of oxalate from crystal soaking is also the same as other oxalate-bound PYK structures from co-crystallization studies [23]. The structural and spectroscopic results presented in the present study suggest that both the decarboxylase and kinase activities of PYKs occur in a common active site and share a mechanism in which the same intermediate enolpyruvate is formed (Figures 7a-7d).…”

“…The B-domain of PYK has been shown to adopt multiple positions in different ligated states, and the position of the B-domain is related to the regulation of kinase activity [13,23]. A large-scale 'in crystallo' B-domain movement with respect to the AC core has been observed by soaking the natural substrate PEP into the crystal of TbPYK-F26BP-Mg resulting in TbPYK-F26BP-PEP-Mg [13].…”

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

“…Recently, it was shown that the allosteric transition between the inactive T state and the active R state involved a symmetrical 6°rigid-body rocking motion of the Aand C-domain cores in each of the four subunits. This also involved the formation of eight essential salt bridge locks across the COC interface that provided tetramer rigidity and a 7-fold increase in the enzyme activity (22).…”

Methicillin-resistant Staphylococcus aureus (MRSA) Pyruvate Kinase as a Target for Bis-indole Alkaloids with Antibacterial Activities

“…The binding pocket was found to be located at the small interface of the PK tetramer and was formed by the anti-parallel interaction of helix 357-370 from the respective subunits ( Structural comparison to eukaryotic homologues provides an explanation for the observed selectivity the bis-indole compounds exhibited toward the S. aureus enzyme. Analysis of the Leishmania mexicana inactive and active structures (13) showed the presence of a similar binding pocket at the small interface, the nature of which changes dependent on the enzymatic state (see below). However, analysis of all the higher eukaryotic structures, regardless of apparent state, showed a much tighter dimeric packing around the small interface, which drastically alters the nature of the pocket, and also the closer interaction of a helix 340 -350 above the interface, thereby limiting access to the pocket (Fig.…”

“…Recently it was shown that the allosteric transition between inactive T-state and active R-state involved a symmetrical 6°rigid-body rocking motion of the Aand C-domain cores in each of the four subunits. This also involved the formation of eight essential salt-bridge locks across the small interface that provided tetramer rigidity and a 7-fold increase in enzyme activity (13). Sequence alignment between MRSA and human PKs revealed particular sequence divergence in domain C (e.g.…”