Structure and Function of Human Erythrocyte Pyruvate Kinase

Valentini, Giovanna; Chiarelli, Laurent R.; Fortin, R.; Dolzan, M.; Galizzi, Alessandro; Abraham, Donald J.; Wang, Changqing; Bianchi, Paola; Zanella, Alberto; Mattevi, Andrea

doi:10.1074/jbc.m202107200

Cited by 130 publications

(85 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The PYK allosteric mechanism has also been investigated using a large number of site point mutants (16 -19). A general conclusion from the wealth of data is that intersubunit interactions on the A-A and C-C interfaces strongly influence the allosteric effect whereas mutations affecting the intrasubunit A-C interface are less sensitive (20).…”

mentioning

confidence: 99%

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

Morgan

McNae

Nowicki

et al. 2010

Journal of Biological Chemistry

131

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 99%

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

Morgan

McNae

Nowicki

et al. 2010

Journal of Biological Chemistry

131

View full text Add to dashboard Cite

show abstract

“…RPK is a 200-kDa tetramer with 4 identical subunits, each consisting of 4 domains. 1 The active site of RPK occurs at a domain interface, and the allosteric site is found in domain C. 2 The PKM gene on chromosome 15 codes for 2 different M-type PK (MPK) forms, M1 (brain and skeletal muscle) and M2 (fetal and most adult tissues), produced by alternative splicing. 3,4 Undifferentiated erythroid precursor cells express the M2PK isoenzyme earlier than mature RBCs express the RPK isoenzyme.…”

mentioning

confidence: 99%

Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression

Díez

Gilsanz

Martínez

et al. 2005

Blood

View full text Add to dashboard Cite

IntroductionPyruvate kinase (PK; EC 2.7.1.40) is a key enzyme of the glycolytic pathway responsible for irreversibly catalyzing the conversion of phosphoenolpyruvate to pyruvate. In humans, pyruvate kinase activity is provided by 4 isoenzymes encoded by 2 structural genes. 1 The PKLR gene on chromosome 1 codes for the R-type PK (RPK) (exclusively in mature red blood cells [RBCs]) and the L-type PK (in liver) isoforms under the control of tissue-specific promoters. RPK is a 200-kDa tetramer with 4 identical subunits, each consisting of 4 domains. 1 The active site of RPK occurs at a domain interface, and the allosteric site is found in domain C. 2 The PKM gene on chromosome 15 codes for 2 different M-type PK (MPK) forms, M1 (brain and skeletal muscle) and M2 (fetal and most adult tissues), produced by alternative splicing. 3,4 Undifferentiated erythroid precursor cells express the M2PK isoenzyme earlier than mature RBCs express the RPK isoenzyme. [5][6][7] Because of the tissue-specific expression patterns of the genes, only mutations in the PKLR gene lead to erythrocyte PK deficiency. 8 PK deficiency severely affects RBC metabolism, causing adenosine triphosphate (ATP) depletion, which ultimately leads to hemolysis. Clinical symptoms vary considerably from mild to severe anemia. For the patient, anemia may mean transfusion dependence and may even be life threatening. Pathologic signs of PK deficiency are usually observed when enzyme activity is less than 25% normal activity and patients are generally homozygotes or compound heterozygotes with 2 different mutant alleles. 9 Most cases of PK deficiency are caused by the production of mutant enzymes with abnormal biochemical properties. Thus far, molecular analyses have identified at least 133 different mutations in the PKLR structural gene. 10 Most are missense mutations, but there are also reports of point mutations, deletions, or insertions that led to more drastic changes, such as alterations of the splicing site, frameshift, early termination mutations, and disruption of erythroid-specific promoters. [10][11][12][13][14] Most mutations constitute single-base substitutions, predicting amino acid changes of conserved residues in structurally and functionally important domains of the RPK tetramer. 15 Mutations that affect PKLR transcription, 14,16 or processing of its premRNA, 13,[17][18][19][20][21][22] are less common. To date, only a few homozygous PK null mutations have been identified. The first reported was the relatively common "PK gypsy," consisting of a large deletion at exon 11 23 with premature termination of its translation losing 10% of the original sequence at the C-terminus (exon 11 is deleted, and 35 aberrant residues are added); afterward, 2 one-base deletions and one transition at a split site 11 were reported. More recently, the first homozygous nonsense mutation was reported, 24 which causes premature termination of translation resulting in a truncated protein lacking a 33-residue C-terminal fragment.Here, we report a novel homozygous PKLR gene...

show abstract

“…Each subunit of PK can be divided into three principal domains: a classic (β/α) 8 barrel A domain, an irregular β-barrel B domain, and a C domain with (α+β) topology. In addition, the N-terminal domain formed by helix-turn-helix motifs of variable length can be identified in mammalian isoenzymes [2,3,[6][7][8]. The active site of PK is located between domains A and B [2][3][4]7], with the binding sites for ADP and PEP involving highly conserved amino acids.…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation is switch of L-type pyruvate kinase allostery

et al. 2010

View full text Add to dashboard Cite

Among four pyruvate kinase isoenzymes, M1, M2, R and L, only M1 is considered as a nonallosteric enzyme. However, here we show that the non-phosphorylated L-type pyruvate kinase (L-PK) is also a non-allosteric enzyme with respect to its substrate phosphoenolpyruvate (PEP). The allosteric catalytic properties of L-PK are switched on through phosphorylation by cAMP-dependent protein kinase. The non-phosphorylated enzyme was produced by expressing the rat L-PK in E. coli, as the bacterium does not have mammalian-type protein kinases. The resulting tetrameric protein was phosphorylated with a stoichiometric ratio of one mole of phosphate per one L-PK monomer. Activity of the phosphorylated enzyme was allosterically regulated by PEP with the Hill coefficient n=2.5. It was observed that allostery was engaged by phosphorylation of the first subunit in the tetrameric enzyme, while further phosphorylation only modulated this effect. The discovered switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating the allostery by phosphorylation are important for understanding of the interrelationship between allostery and the regulatory phosphorylation in general, and may have implication for further analysis of glycolysis regulation in the liver.© Versita Sp. z o.o. Keywords: Regulatory phosphorylation • Allostery • L-type pyruvate kinase • Switch between allosteric and non-allosteric forms • Glycolysis regulation

show abstract

Structure and Function of Human Erythrocyte Pyruvate Kinase

Cited by 130 publications

References 37 publications

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

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

Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression

Phosphorylation is switch of L-type pyruvate kinase allostery

Contact Info

Product

Resources

About