Postnatal changes in canine erythrocyte pyruvate kinase isozymes

The isozyme pattern of pyruvate kinase in canine erythrocytes changes following birth. These changes have been followed by electrophoretic, immunologic, and kinetic measurements of the isozymes. At birth, a mixture of isozymes is present consisting of the M₂ isozyme and hybrid molecules containing M₂ and R subunits. With increasing animal age, the content of M₂ subunits decreases and the content of R subunits increases. At 6 months of age, the isozyme pattern is indistinguishable from that of adult erythrocytes which contain only the R tetramer. We conclude that there is a switch in erythrocyte pyruvate kinase gene expression during the first 6 months of postnatal life. The existence of hybrid molecules during the switch indicates that both M₂ and R genes are expressed within each erythroid precursor cell. The developmental changes in erythrocyte pyruvate kinase are consistent with the role of this enzyme in the regulation of the oxygen-transport function of canine hemoglobin by 2,3-diphosphoglycerate in the postnatal period.This research was supported by Public Health Service Grant HD-10595.     

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L fromRana pipiens

Summary The regulatory properties of type L pyruvate kinase fromRana pipiens are intermediate between those of the mammalian K and L isozymes. As with mammalian type L, the levels of the frog isozyme are affected by the animal's nutritional state. The mammalian and amphibian isozymes show similar sensitivities to fructose 1,6-bisphosphate activation and amino acid inhibition. By contrast, the frog L isozyme shares several properties of the K class: ie. irreversible inactivation by oxidized glutathione and lack of response to a cyclic AMP stimulated phosphorylation. Furthermore, as for some mammalian K isozymes, frog type L shows a high PEP affinity and a low cooperativity of PEP binding.Insofar as the properties of this present day enzyme reflect those of its counterpart in the amphibian ancestor of higher vertebrates, our results suggest that at its first expression, the type L resembled the type K. Many important regulatory properties of the L isozyme, especially the sensitivity to phosphorylation, were acquired more recently perhaps in association with an increased importance of constant blood glucose.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N-tetraacetic acid - FB fructose-1,6-bisphosphate - PEP phosphoenolpyruvate - PK pyruvate kinase     

Differentiating a ligand's chemical requirements for allosteric interactions from those for protein binding. Phenylalanine inhibition of pyruvate kinase

The isoform of pyruvate kinase from brain and muscle of mammals (M(1)-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the l-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M(1)-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the beta-carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the beta-carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 A structure of M(1)-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the l-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.     

Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis

Four isozymes of pyruvate kinase are differentially expressed in human tissue. Human pyruvate kinase isozyme M2 (hPKM2) is expressed in early fetal tissues and is progressively replaced by the other three isozymes, M1, R, and L, immediately after birth. In most cancer cells, hPKM2 is once again expressed to promote tumor cell proliferation. Because of its almost ubiquitous presence in cancer cells, hPKM2 has been designated as tumor specific PK-M2, and its presence in human plasma is currently being used as a molecular marker for the diagnosis of various cancers. The X-ray structure of human hPKM2 complexed with Mg(2+), K(+), the inhibitor oxalate, and the allosteric activator fructose 1,6-bisphosphate (FBP) has been determined to a resolution of 2.82 A. The active site of hPKM2 is in a partially closed conformation most likely resulting from a ligand-induced domain closure promoted by the binding of FBP. In all four subunits of the enzyme tetramer, a conserved water molecule is observed on the 2-si face of the prospective enolate and supports the hypothesis that a proton-relay system is acting as the proton donor of the reaction (1). Significant structural differences among the human M2, rabbit muscle M1, and the human R isozymes are observed, especially in the orientation of the FBP-activating loop, which is in a closed conformation when FBP is bound. The structural differences observed between the PK isozymes could potentially be exploited as unique structural templates for the design of allosteric drugs against the disease states associated with the various PK isozymes, especially cancer and nonspherocytic hemolytic anemia.     

Mining for allosteric information: natural mutations and positional sequence conservation in pyruvate kinase

Although the amino acid sequences and the structures of pyruvate kinase (PYK) isozymes are highly conserved, allosteric regulations differ. This suggests that amino acids with low conservation play important roles in the allosteric mechanism. The current work exploits a 'natural screen'- the 122 point mutations identified in the human gene encoding the erythrocyte PYK isozyme and associated with nonspherocytic hemolytic anemia - to learn what amino acid positions in PYK may be important for allosteric regulations. In addition to the mutations, we consider the conservation of each amino acid position across 241 PYK sequences. Three groups of residue positions have been created, those with: (1) no disease causing mutation identified; (2) a disease causing mutation identified and high conservation across isozymes; and (3) a disease causing mutation identified and low conservation. Mutations at positions not identified in the natural screen are likely to be tolerated with minimal loss of function. Mutations at highly conserved positions are more likely to disrupt properties common to all PYK isozymes (e.g., structure, catalysis). Residues in the third group are likely to be involved in roles that are necessary for function but not common to all isozymes (e.g., allostery). Many of the Group 3 residues are located in the C-domain and to a lesser extent the A domain.     

Inherited persistence of immature type pyruvate kinase and hexokinase isozymes in dog erythrocytes

1. Red cell pyruvate kinase (EC 2.7.1.40) and hexokinase (EC 2.7.1.1) in high and low potassium (K) dogs were shown to exist as multiple forms which were separable by electrophoresis and ion-exchange chromatography. The R2-type pyruvate kinase, which was determined to be a young type enzyme in canine red cells, was shown to be the predominant form of pyruvate kinase in high K cells. 2. The M2-type pyruvate kinase, a prototype isozyme in erythroid cells, existed in high K dog erythrocytes as well as in high K and low K dog reticulocytes. 3. Isozyme analysis of high K red cell hexokinase also showed a profile similar to that obtained for low K reticulocytes. 4. These results seem to reflect the immaturity of high K erythrocytes, which suggest that an abnormal cell differentiation or maturation may occur at an early stage of erythroid cell proliferation in high K dogs.     

An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas.

1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.     

A network of conserved interactions regulates the allosteric signal in a glutamine amidotransferase

We have combined equilibrium and steered molecular dynamics (SMD) simulations with principal component and correlation analyses to probe the mechanism of allosteric regulation in imidazole glycerol phosphate (IGP) synthase. An evolutionary analysis of IGP synthase revealed a conserved network of interactions leading from the effector binding site to the glutaminase active site, forming conserved communication pathways between the remote active sites. SMD simulations of the undocking of the ribonucleotide effector N1-[(5'-phosphoribulosyl)-formino]-5'-aminoimidazole carboxamide ribonucleotide (PRFAR) resulted in a large scale hinge-opening motion at the interface. Principal component analysis and a correlation analysis of the equilibration protein motion indicate that the dynamics involved in the allosteric transition are mediated by coupled motion between sites that are more than 25 A apart. Furthermore, conserved residues at the substrate-binding site, within the barrel, and at the interface were found to exhibit highly correlated motion during the allosteric transition. The coupled motion between PRFAR unbinding and the directed opening of the interface is interpreted in combination with kinetic assays for the wild-type and mutant systems to develop a model of allosteric regulation in IGP synthase that is monitored and investigated with atomic resolution.     

Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes.

Pyruvate kinase isozymic changes were studied in the adult hepatocyte cultures, by electrophoretic, kinetic and immunological methods. We were able to maintain parenchymal cells from normal adult rat liver in non-proliferating monolayer cultures up to 10 days. Hepatocytes appeared to contain a dominant PK I type up to 4-5 days of culture. After day 5, PK III type was regularly present with PK I and after 7 days PK III type was always the only isozyme detected in culture. It must be pointed out that, by the Ouchterlony method and sometimes by electrophoresis, concentrated extracts from freshly isolated hepatocytes or starting hepatocyte cultures did also contain Pyruvate kinase PK III type. These results suggest that Pyruvate kinase III is present but partly repressed in the adult parenchymal cells and becomes derepressed in culture.     

Evolutionary variation and adaptation in a conserved protein kinase allosteric network: Implications for inhibitor design

The activation of protein kinases involves conformational changes in key functional regions of the kinase domain, a detailed understanding of which is essential for the design of selective protein kinase inhibitors. Through statistical analysis of protein kinase sequences and crystal structures from diverse organisms, we recently proposed that the activation of protein kinases involves a hidden strain switch in the catalytic loop. Specifically, we demonstrated that the backbone torsion-angles of residues in the catalytic loop switch from a “relaxed” to “strained” conformation upon kinase activation and the strained geometry results in a network of hydrogen bonds involving conserved non-catalytic residues in the ATP and substrate binding lobes. Here, we further explore this activation mechanism by analyzing families that lack the canonical hydrogen bonding interactions with the strained backbone. We find that alternative mechanisms have evolved to maintain catalytic loop strain. In PIM kinase, for example, two water molecules account for the lack of a conserved aspartate in the substrate binding by hydrogen bonds to the strained backbone. We discuss the relevance of these findings in the design of family-specific allosteric inhibitors, and in predicting the structural and functional impact of cancer mutations that alter the strain associated hydrogen bonding network. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

New roles for pyruvate kinase M2: working out the Warburg effect

The origins and role of the Warburg effect have remained uncertain for many years. Two recent studies demonstrate that an embryonic- and cancer-cell-specific isoform of the enzyme pyruvate kinase M2 (PKM2) is regulated by binding to phospho-tyrosine motifs and promotes increased cell growth and tumor development. PKM2 enhances the use of glycolytic intermediates for macromolecular biosynthesis and tumor growth. These findings illustrate the distinct advantages of this metabolic phenotype in cancer cell growth.     

Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase.

The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A [bond] A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A [bond] A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C [bond] C interface only moderately influenced allostery. Creating an E392A mutation in the C [bond] C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A [bond] C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.     

Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching

Pyruvate kinase acts as an allosteric enzyme, playing a crucial role in the catalysis of the final step of the glycolytic pathway. In this study, site-specific mutagenesis and tryptophan fluorescence quenching were used to probe the catalytic allosteric mechanism of rabbit muscle pyruvate kinase. Movement of the B domain was found to be essential for the catalytic reaction. Rotation of the B domain in the opening of the cleft between domains B and A induced by the binding of activating cations allows substrates to bind, whereas substrate binding shifts the rotation of the B domain in the closure of the cleft. Trp-157 accounts for the differences in tryptophan fluorescence signal with and without activating cations and substrates. Trp-481 and Trp-514 are brought into an aqueous environment after phenylalanine binding.     

Dominant negative role of the glutamic acid residue conserved in the pyruvate kinase M(1) isozyme in the heterotropic allosteric effect involving fructose-1,6-bisphosphate

Pyruvate kinase M(1), a nonallosteric isozyme, lacks heterotropic allosteric effect involving fructose-1,6-bisphosphate (FBP). To explore the molecular basis for this, a series of mutants were prepared and characterized, in which the possible candidate, Glu-432, was replaced in the rat M(1) isozyme and its allosteric mutant with the replacement of Ala-398 by Arg. Although these single mutants of Glu-432 remained nearly fully active, similar to the wild type, only the mutants with replacements by Lys and Ala were more efficiently activated by FBP when the enzymes were inhibited by L-phenylalanine. Kinetic analyses and ligand-induced fluorescence quenching studies using the allosteric double mutants indicated that the loss of a negative charge at residue 432 led to a dramatic decrease in the apparent activation constant and apparent K(d) for FBP. Furthermore, this enhancement was found to be associated with the modification of the FBP-binding site rather than the alteration of the subunit assembly. These findings suggest that Glu-432 hinders the heterotropic allosteric effect by preventing the binding of FBP through a repulsive electrostatic interaction and thereby contributes to its unique unregulated properties, independent of the shifted allosteric transition.     

Comparative homology modeling of pyruvate dehydrogenase kinase isozymes from Xenopus tropicalis reveals structural basis for their subfunctionalization

Structural-functional divergence is responsible for the preservation of highly homologous genes. Protein functions affected by mutagenesis in divergent sequences require investigation on an individual basis. In the present study, comparative homology modeling and predictive bioinformatics analysis were used to reveal for the first time the subfunctionalization of two pyruvate dehydrogenase kinase (PDK) isozymes in the western clawed frog Xenopus tropicalis. Three-dimensional structures of the two proteins were built by homology modeling based on the crystal structures of mammalian PDKs. A detailed comparison of them revealed important structural differences that modify the accessibility of the nucleotide binding site in the two isozymes. Based on the generated models and bioinformatics data analysis, the differences between the two proteins in terms of kinetic parameters, metabolic regulation, and tissue distribution are predicted. The results obtained are consistent with the idea that one of the xtPDKs is the major isozyme responsible for metabolic control of PDC activity in X. tropicalis, whereas the other one has more specialized functions. Hence, this study provides a rationale for the existence of two closely related PDK isozymes in X. tropicalis, thereby enhancing our understanding of the functional evolution of PDK family genes.     

Hybrid isozymes of rat pyruvate kinase. Their subunit structure and developmental changes in the liver

Thin-layer polyacrylamide gel electrophoresis of various rat tissues revealed three major isozymes (types L, M1 and M2) and various intermediate forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40). In vitro dissociation and reassociation of purified enzymes showed that the three major isozymes had homotetrameric structures. L.M2 hybrids and M1.M2 hybrids closely resembled some naturally occurring intermediates; the subunit structure of intermediates isolated from the small intestine (form 3 or form 4) were estimated to be (L)2(M2)2 and (L)(M2)3, respectively. Pyruvate kinase activity after electrophoresis could be estimated quantitatively from densitometric measurements of the electrophoretic pattern. Type L activity in fetal liver was separated from type R activity derived from intrahepatic erythropoietic cells. It changes in three distinct steps during development: it increased during the late fetal period, remained steady during the neonatal period and increased again after weaning. Some of the intermediates found in extracts of early fetal iver were shown to cross-react with both anti-L and anti-M1 serum, suggesting that they might be L.M2 or R.M2 hybrids. These hybrid enzymes were shown to appear only during early fetal and neonatal periods.     

Key residues in the allosteric transition of Bacillus stearothermophilus pyruvate kinase identified by site-directed mutagenesis.

The structural gene for pyruvate kinase from Bacillus stearothermophilus has been cloned in Escherichia coli and sequenced. The open reading frame from the ATG start codon to the TAG stop codon is 1482 base-pairs and encodes a peptide of relative molecular mass 52,967. In the expression vector pKK223-3, containing the synthetic tac promoter, the gene is overexpressed in E. coli cells to an estimated level of 30% total soluble cell protein. A purification procedure for the overexpressed protein has been established. The construction and characterization of a pair of mutant proteins has given insight into the structural basis of allosteric regulation in the tetrameric enzyme. Substituting tryptophan for tyrosine at position 466 (mutant Trp466-->Tyr) resulted in an activated form of the enzyme, having a reduced K1/2 for the substrate phosphoenolpyruvate. We propose that the characteristics of this mutant might be the result of bulk removal releasing steric inhibition to the formation of an interdomain salt bridge between Asp356 and Arg444. The regulatory behaviour of the double mutant produced by making the additional substitution aspartate for glutamate at position 356 (Trp466-->Tyr/Asp356-->Glu) corroborates this. The position of the salt bridge is such that it might be pivotal to the conformation of a pocket that is proposed to open up when the active R-conformation is adopted. We suggest that the mechanism of activation of B. stearothermophilus pyruvate kinase by ribose-5-phosphate might hinge on an interaction with, or indirectly through, residue Trp466, removing it from the vicinity of the potential salt bridge between Asp356 and Arg444 and thus effecting a closing together of the protein structure concomitant with an opening up of the pocket region.