Mammalian Pyruvate Kinase Research Articles

The pyruvate kinase of the whiteleg shrimp Litopenaeus vannamei: Gene structure and responses to short term hypoxia

The shrimp Litopenaeus vannamei is the main farmed crustacean worldwide. This shrimp suffers environmental changes in oxygen availability that affect its energy metabolism. Pyruvate kinase (PK) catalyzes the last reaction of glycolysis and is key for the regulation of glycolysis and gluconeogenesis. There is ample knowledge about mammalian PK, but in crustaceans, the information is very scarce. In this study, we analyzed in silico the structures of the PK gene and protein. Also, the effects of hypoxia on gene expression, enzymatic activity, glucose, and lactate in hepatopancreas and muscle were analyzed. The PK gene is 15,103 bp and contains 11 exons and 10 introns, producing four mRNA variants by alternative splicing and named PK1, PK2, PK3 and PK4, that results in two proteins with longer C-terminus and two with a 12 bp insertion. The promoter contains putative binding sites for transcription factors (TF) that are typically involved in stress responses. The deduced amino acid sequences contain the classic domains, binding sites for allosteric effectors and potential reversible phosphorylation residues. Protein modeling indicates a homotetramer with highly conserved structure. The effect of hypoxia for 6 and 12 h showed tissue-specific patterns, with higher expression, enzyme activity and lactate in muscle, but higher glucose in hepatopancreas. Changes in response to hypoxia were detected at 12 h in expression with induction in muscle and reduction in hepatopancreas, while enzyme activity was maintained, and glucose and lactate decreased. These results show rapid changes in expression and metabolites, while enzyme activity was maintained to cope with short-term hypoxia.

Cancer-associatedmutations in human pyruvate kinase M2 impair enzyme activity.

Mammalian pyruvate kinase catalyzes the final step of glycolysis, and its M2 isoform (PKM2) is widely expressed in proliferative tissues. Mutations in PKM2 are found in some human cancers; however, the effects of these mutations on enzyme activity and regulation are unknown. Here, we characterized five cancer-associated PKM2 mutations, occurring at various locations on the enzyme, with respect to substrate kinetics and activation by the allosteric activator fructose-1,6-bisphosphate (FBP). The mutants exhibit reduced maximal velocity, reduced substrate affinity, and/or altered activation by FBP. The kinetic parameters of five additional PKM2 mutants that have been used to study enzyme function or regulation also demonstrate the deleterious effects of mutations on PKM2 function. Our findings indicate that PKM2 is sensitive to many amino acid changes and support the hypothesis that decreased PKM2 activity is selected for in rapidly proliferating cells.

Structures of pyruvate kinases display evolutionarily divergent allosteric strategies.

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (kcat/S0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (kcat/S0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.

Concentration-dependent control of pyruvate kinase M mutually exclusive splicing by hnRNP proteins

Expression of the mammalian pyruvate kinase M (PKM) gene provides an important example of mutually exclusive splicing. We showed previously that the hnRNP proteins A1, A2 and PTB have a crucial role in this process. Here we provide evidence that concentration-dependent interactions involving a network of these proteins are sufficient to determine the outcome of PKM splicing. At high concentrations, such as those found in most cancer cells, hnRNPA1 binding to two sites in the upstream regulated exon (exon 9) orchestrates cooperative interactions leading to exon 9 exclusion. At lower concentrations, binding shifts to downstream intronic sites, such that exon 9 is included and exon 10 mainly excluded, with any mRNA including both exons degraded by nonsense-mediated decay. Together, our results provide a mechanism by which a few general factors control alternative splicing of a widely expressed transcript.

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 3. Mechanism

Mammalian pyruvate kinase exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues. All of the isoforms, except the muscle isoform, exhibit typical allosteric behavior. The case of the muscle isoform is a conundrum. It is inhibited by an allosteric inhibitor, Phe, yet it has traditionally not been considered as an allosteric enzyme. In this series of study, an energetic landscape of rabbit muscle pyruvate kinase (RMPK) was established. The phenomenon of inhibition by Phe is shown to be physiological. Furthermore, the thermodynamics for the temperature fluctuation and concomitant pH change as a consequence of muscle activity were elucidated. We have shown that (1) the differential number of protons released or absorbed with regard to the various linked reactions adds another level of control to shift the binding constants and equilibrium of active <--> inactive state changes (the latter controls quantitatively the activity of RMPK); (2) ADP plays a major role in the allosteric mechanism in RMPK under physiological temperatures (depending on the temperature, ADP can assume dual and opposite roles of being an inhibitor by binding preferentially to the inactive form and a substrate); and (3) simulation of the RMPK behavior under physiological conditions shows that the net results of the 21 thermodynamic parameters involved in the regulation are well-tuned to allow the maximal response of the enzyme to even minute changes in temperature and ligand concentration.

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM) (alpha/beta)(8) barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (alpha/beta)(8) structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E(T) and active E(R). These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E(T) and active E(R) represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.

Pyruvate kinase type M2: a crossroad in the tumor metabolome

Cell proliferation is a process that consumes large amounts of energy. A reduction in the nutrient supply can lead to cell death by ATP depletion, if cell proliferation is not limited. A key sensor for this regulation is the glycolytic enzyme pyruvate kinase, which determines whether glucose carbons are channelled to synthetic processes or used for glycolytic energy production. In unicellular organisms pyruvate kinase is regulated by ATP, ADP and AMP, by ribose 5-P, the precursor of the nucleic acid synthesis, and by the glycolytic intermediate fructose 1,6-P2 (FBP), thereby adapting cell proliferation to nutrient supply. The mammalian pyruvate kinase isoenzyme type M2 (M2-PK) displays the same kinetic properties as the pyruvate kinase enzyme from unicellular organisms. The mammalian M2-PK isoenzyme can switch between a less active dimeric form and a highly active tetrameric form which regulates the channeling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumor cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. The tetramer-dimer ratio is regulated by ATP, FBP and serine and by direct interactions with different oncoproteins (pp60v-src, HPV-16 E7). In solid tumors with sufficient oxygen supply pyruvate is supplied by glutaminolysis. Pyruvate produced in glycolysis and glutaminolysis is used for the synthesis of lactate, glutamate and fatty acids thereby releasing the hydrogen produced in the glycolytic glyceraldehyde 3-phosphate dehydrogenase reaction.

Structural and Functional Linkages Between Subunit Interfaces in Mammalian Pyruvate Kinase

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8Å. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Nutrient and hormonal regulation of pyruvate kinase gene expression

Mammalian pyruvate kinase (PK), a key glycolytic enzyme, has two genes named PKL and PKM, which produce the L- and R-type isoenzymes by means of alternative promoters, and the M1-and M2-types by mutually exclusive alternative splicing respectively. The expression of these genes is tissue-specific and under developmental, dietary and hormonal control. The L-type isoenzyme (L-PK) gene contains multiple regulatory elements necessary for regulation in the 5' flanking region, up to position -170. Both L-II and L-III elements are required for stimulation of L-PK gene transcription by carbohydrates such as glucose and fructose, although the L-III element is itself responsive to carbohydrates. The L-II element is also responsible for the gene regulation by polyunsaturated fatty acids. Nuclear factor-1 proteins and hepatocyte nuclear factor 4, which bind to the L-II element, may also be involved in carbohydrate and polyunsaturated fatty acid regulation of the L-PK gene respectively. However, the L-III-element-binding protein that is involved in carbohydrate regulation remains to be clarified, although involvement by an upstream stimulating factor has been proposed. Available evidence suggests that the carbohydrate signalling pathway to the L-PK gene includes a glucose metabolite, possibly glucose 6-phosphate or xylulose 5-phosphate, as well as phosphorylation and dephosphorylation mechanisms. In addition, at least five regulatory elements have been identified in the 5' flanking region of the PKM gene up to position -279. Sp1-family proteins bind to two proximal elements, but the binding of proteins to other elements have not yet been clarified. Glucose may stimulate the transcription of the PKM gene via hexosamine derivatives. Sp1 may be involved in this regulation via its dephosphorylation, although the carbohydrate response element has not been determined precisely in the PKM gene. Thus glucose stimulates transcription of the PKM gene by the mechanism which is probably different from the L-PK gene.

The Negative Dominant Effects of T340M Mutation on Mammalian Pyruvate Kinase

A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.

Purification of a novel pyruvate kinase from a green alga

A chloroplast pyruvate kinase has been purified to homogeneity from the green alga Selenastrum minutum. The subunit composition of the enzyme differs markedly from pyruvate kinases from other sources. Subunit and native M rs were 210 kDa and 235 kDa, respectively, indicating a monomeric structure. Immunological and peptide mapping analyses suggested structural homology to a bacterial, but not higher plant or mammalian pyruvate kinase.

Structure of the Aspergillus nidulans pyruvate kinase gene.

The complete nucleotide sequence of the Aspergillus nidulans pyruvate kinase gene, including its flanking sequences, is presented. The gene has a 1,578 bp coding sequence that encodes a protein of 526 amino acids; the latter is strongly homologous to the pyruvate kinases found in Saccharomyces cerevisiae (66%) and mammals (53%). The gene is interrupted by seven introns, three of which are in a conserved position compared to those present in the mammalian pyruvate kinase genes sequenced thus far. A fourth intron within the mononucleotide binding fold domain is in a conserved position with respect to the position of an intron within the NAD+ binding region of maize ADH I. The transcription start site has been determined; a major site of transcription was found 80 bp before the translation initiation codon. The promoter region of the A. nidulans pyruvate kinase gene contains no direct homologies with the TATA or CCAAT sequences in the expected region (30-70 bp) before the transcription initiation site. However, extended CT-enriched regions are found in the promotor region, similar to what has been observed in genes that are highly expressed in Saccharomyces cerevisiae and filamentous fungi.

Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis

1. 1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with K m s for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a K m of about 0.15 mM; the kinetics become hyperbolic and the K m for PEP is reduced to 0.05 mM in the presence of μM concentrations of fructose-1,6-bisphosphate. 4. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.

A study of the catalytic properties of pyruvate kinase isozymes from salmon and an examination of their functional relationships

AbstractTwo major isozymes of pyruvate kinase that differ in electrophoretic and kinetic properties are found in salmon (Oncorhynchus kisutch). Type M occurs in cardiac and skeletal muscle, while type K is the main isozyme of the other tissues. We have been examining the structural and kinetic properties of the pyruvate kinase isozymes of salmon in an effort to assess their regulatory properties as well as their structural and functional relationships to the isozymes from “higher” vertebrates. We find that type K pyruvate kinase has hyperbolic kinetics with both phosphoenolpyruvate and ADP but that the apparent affinities for the two substrates are interdependent. That is, decreased affinities for phosphoenolpyruvate occur at higher concentrations of ADP and vice versa. In contrast, salmon type M pyruvate kinase has slightly sigmoidal kinetics with phosphoenolpyruvate, indicative of cooperative interactions among subunits of the latter isozyme. However, the affinity of this isozyme for each substrate is much less effected by the concentration of the other substrate. Salmon type K pyruvate kinase binds Mg++ cooperatively in the absence of fructose bisphosphate, where as type M binds Mg++ twice as tightly but in a non cooperative fashion. Both enzymes are inhibited to a similar extent by ATP, but they may differ in the nature of this inhibition. The salmon isozymes differ from avian and mammalian pyruvate kinase in that they are much less susceptible to inhibition by alanine and phenylalanine. The kinetic properties of salmon type K pyruvate kinase imply it to be more amenable to regulation that salmon type M, a situation that is also true for the corresponding isozymes from birds and mammals. Nevertheless, the two salmon isozymes appear to be more similar kinetically to each other than are types K and M pyruvate kinases from higher vertebrates, suggesting less evolutionary divergence for the salmon isozymes.

Maximum activities and effects of fructose bisphosphate on pyruvate kinase from muscles of vertebrates and invertebrates in relation to the control of glycolysis

1. Comparison of the maximum activities of pyruvate kinase with those of phosphofructokinase in a large number of muscles from invertebrates and vertebrates indicates that, in general, in any individual muscle, the activity of pyruvate kinase is only severalfold higher than that of phosphofructokinase. This is consistent with the suggestion, based on mass-action ratio data, that the pyruvate kinase reaction is non-equilibrium in muscle. However, the range of activities of pyruvate kinase in these muscles is considerably larger than that of phosphofructokinase. This difference almost disappears if the enzyme activities from muscles that are known to possess an anaerobic ;succinate pathway' are excluded. It is suggested that, in these muscles, phosphofructokinase provides glycolytic residues for both pyruvate kinase (i.e. glycolysis) and phosphoenolpyruvate carboxykinase (i.e. the succinate pathway). This is supported by a negative correlation between the activity ratio, pyruvate kinase/phosphofructokinase, and the activities of nucleoside diphosphokinase in these muscles, since high activities of nucleoside diphosphokinase are considered to indicate the presence of the succinate pathway. 2. The effect of fructose bisphosphate on the activities of pyruvate kinase from many different muscles was studied. The stimulatory effect of fructose bisphosphate appears to be lost whenever an efficient system for supply of oxygen to the muscles is developed (e.g. insects, squids, birds and mammals). This suggests that activation of pyruvate kinase is important in the co-ordinated regulation of glycolysis in anaerobic or hypoxic conditions, when the change in glycolytic flux during the transition from rest to activity needs to be large in order to provide sufficient energy for the contractile activity. However, lack of this effect in the anaerobic muscles of the birds and mammals suggests that another metabolic control may exist for avian and mammalian pyruvate kinase in these muscles.

Bovine pyruvate kinase isozymes and hybrid isozymes: Electrophoretic studies and tissue distribution

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K 4, L 4, and M 4), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K 2L 2 and K 3M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K 4 also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M 4. The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M 4 with a little KM 3. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K 3M, while Purkinje fibers have a five-membered set dominated by KM 3. The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.

A comparative electrophoretic analysis of mammalian pyruvate kinase isozymes

1.1. A complex system of four major pyruvate kinase (PK) isozymes has been demonstrated in the tissues of rat, mouse, guinea pig, rabbit and sheep.2.2. In each of these animals a marked uniformity was shown with respect to the tissue distribution and specific activity of each isozyme: PK-A4 in muscle, heart and brain; PK-B4 in liver and kidney; PK-C4 in kidney, spleen and testis; and erythrocyte PK.3.3. Comparative electrophoretic results of erythrocyte PK together with previously reported immunochemical properties for this isozyme (Imamura & Tanaka, 1972), strongly suggest a PK-B3C subunit structure.4.4. Sets of PK B/C subunit hybrid isozymes were consistently observed in the livers and kidneys of rat and mouse as well as in kidneys of rabbit, guinea pig and sheep. An A/C subunit PK hybrid isozyme set was also identified in rat testis and spleen.5.5. Electrophoresis of rabbit PK isozymes at neutral and alkaline pH's resulted in a marked change in the migration of PK-C4 in comparison to the other isozymes.6.6. Sheep kidney exhibited mostly PK-B4 activity whereas guinea pig, mouse, and rat kidney contained predominantly PK-C4.

A comparative study of trypanosome and mammalian pyruvate kinase and the action of arsenical drugs

A comparative study of trypanosome and mammalian pyruvate kinase and the action of arsenical drugs