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PubMed1995

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds.

Plastid pyruvate kinase (PKp) activity and anti-(castor oil seed [COS] PKp) immunoglobulin G immunoreactive polypeptides were recovered in the stroma but not from envelope membranes of purified COS leukoplasts that had been subfractionated by sucrose density gradient centrifugation. The PKp was highly purified from isolated leukoplasts using anion-exchange and ADP-agarose chromatographies. Proteolysis of PKp was almost entirely eliminated by including 2,2[prime]-dipyridyl disulfide in purification buffers. The final preparation contained 63.5-kD ([alpha] subunit) and 54-kD ([beta] subunit) polypeptides that stained for protein and cross-reacted with anti-(COS PKp) immunoglobulin G with similar intensities. These two polypeptides co-eluted following gel-filtration chromatography and co-migrated during nondenaturing isoelectric focusing-polyacrylamide gel electrophoresis. The enzyme's native Mr was estimated to be 334,000. This PKp thus appears to exist as an [alpha]3[beta]3-heterohexamer. Comparison of the respective N-terminal sequences of the [alpha] and [beta] subunits with the deduced amino acid sequences for several PKp cDNAs indicated that (a) the [alpha] and [beta] subunits are encoded by COS genes previously designated as PKpA and PKpG, respectively, and (b) respective transit peptides of 4.8- and 5.5-kD are cleaved from the [alpha] and [beta] subunit preproteins following their translocation into the leukoplast.

Journal Article↗
PubMed1976

Studies on the mechanism and stereochemical properties of the oxalacetate decarboxylase activity of pyruvate kinase.

When cod fish muscle oxalacetate decarboxylase catalyzes the decarboxylation of oxalacetate in the presence of NaBH4, L-lactate results from the reduction of enzyme-bound pyruvate. However, D-lactate results when borohydride reduces the binary enzyme-pyruvate complex formed by adding pyruvate from solution, as reported by others. This observation suggests that there are alternate mechanisms for reduction that are either kinetically or sterically determined for the E-pyruvate forms produced in the two directions. In the process of investigating the mechanism of reduction, the cod fish muscle decarboxylase was discovered to be identical with pyruvate kinase. Decarboxylase activity appears to take place at a site which overlaps the phosphoenolpyruvate binding site on this enzyme, as discussed in the following paper. Crystalline rabbit muscle pyruvate kinase also contains significant decarboxylase activity indicating that the two reactions may be structurally related functions. In the presence of K+, orthophosphate, or ATP the rabbit muscle enzyme catalyzes the detritiation of enzyme-bound pyruvate formed during decarboxylation before release of pyruvate from the enzyme, in analogy with the detritiation of pyruvate formed from P-[3-3/]enolpyruvate in the kinase reaction. This observation is consistent with the formation of an enolpyruvate intermediate common to the kinetic pathways of both reactions. Since the decarboxylase reac.tion is completely stereospecific, within the limits of detection, going with retention of configuration, the protonation of the enolpyruvate intermediate is completely determined by the enzyme as is the case with the enolpyruvate intermediate generated from P-enolpyruvate in the kinase reaction.

Animals↗
PubMed1995

Molecular cloning of the genes for pyruvate kinase of two bacilli, Bacillus psychrophilus and Bacillus licheniformis, and comparison of the properties of the enzymes produced in Escherichia coli.

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by ATP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.

Amino Acid Sequence↗
PubMed1977

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.

Adenine Nucleotides↗
PubMed1999

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.

Bacillus subtilis↗
PubMed1990

Regulation of L-type pyruvate kinase gene expression by dietary fructose in normal and diabetic rats.

Previous studies have shown that dietary fructose stimulates expression of the L-type pyruvate kinase gene mainly at the post-transcriptional level in diabetic liver, and that this effect may be mediated by a metabolite common to both fructose and glycerol. In the present work, we carried out further studies on the mechanism of fructose induction of L-type isozyme mRNA in the liver, kidney and small intestine of rats. The L-type isozyme mRNA in the kidney of normal and diabetic rats was increased by dietary fructose, the time course of the increase being similar to that observed in the small intestine and liver. The mRNA was not induced in the kidney by dietary glucose or insulin. Glycerol was also a potent inducer of the mRNA in the kidney and liver, but not in the small intestine. These results show that fructose and glycerol induced increase in the mRNA only in organs in which they were metabolized, and thus support the metabolite hypothesis. No other carbohydrates tested increased the level of mRNA in these tissues, except glucose, which increased the level in the small intestine. Thus a molecule that increase the mRNA level may accumulate significantly during metabolism of only certain carbohydrates. Dietary fructose or glycerol slightly stimulated transcription of the gene for the L-type isozyme in diabetic liver, and also in normal and diabetic kidney, but the magnitudes of increase were much lower than those of the mRNA, confirming our previous findings described above. On the other hand, dietary fructose caused marked stimulation of gene transcription in normal rat liver, although the magnitude of its induction of the mRNA was similar to that in diabetic liver. Insulin treatment of fructose-fed diabetic rats also caused a marked increase in the gene transcription without any concomitant change in the mRNA level. Thus, the mechanism of fructose induction of the L-type pyruvate kinase in diabetic liver, which is similar to that found in the kidney, is different from that in normal liver, and this difference is attributable to the difference in the level of insulin.

Animals↗
PubMed1991

Enolpyruvate: chemical determination as a pyruvate kinase intermediate.

Despite many studies suggesting the role of enolpyruvate as a bound intermediate in the pyruvate kinase reaction, direct evidence for it has been lacking. By use of a combination of chemical trapping and isolation of a derivative, significant amounts of enzyme-bound enolpyruvate have now been demonstrated. The method distinguishes enolpyruvate. It is based on reaction of bromine with enolpyruvate in acid, derivatization of formed bromopyruvate with thionitrobenzoate, and resolution by reversed-phase HPLC of the thioether derivative. As little as 10 pmol of the thioether derivative could be quantitated reliably. With this method, the internal equilibria, including the E.ATP.enolpyruvate intermediate, have been determined. Enzyme-enolpyruvate concentration was shown to be pH-dependent. Phosphoenolpyruvate also reacts with bromine to form bromopyruvate. To quantitate enolpyruvate specifically in a background of phosphoenolpyruvate, advantage was taken of phosphoenolpyruvate's much greater stability in acid. When bromide/was added 10 min after the acid quench, ketonization of enolpyruvate was complete, and only phosphoenolpyruvate was measured. Enolpyruvate is thus determined by difference between the bromopyruvate measured with and without delayed bromine addition.

Adenosine Triphosphate↗
PubMedDate not supplied

The ATP level and pyruvate kinase activity in dog red cells.

The ATP level and pyruvate kinase (PK) activity were measured in red cells of 21 dogs. The obtained results are: ATP -- 732 +/- 197.3 mumoles/1 RBC, PK -- 919 +/- 452.3 mumoles/min/1 RBC. The ATP level in dog red cells was significantly lower than in rabbit and rat red cells (p less than 0.05). Possible explanation for the low ATP level in dog red cells is discussed.

Adenosine Triphosphate↗
PubMed2000

Altered regulation of pyruvate kinase or co-overexpression of phosphofructokinase increases glycolytic fluxes in resting Escherichia coli.

Glycolytic fluxes in resting Escherichia coli were enhanced by overexpression of heterologous pyruvate kinases (Pyk) from Bacillus stearothermophilus and Zymomonas mobilis, but not homologous Pyk. Compared to the control, an increase of 10% in specific glucose consumption and of 15% in specific ethanol production rates was found in anaerobic resting cells, expressing the heterologous Pyks, that were harvested from exponentially growing aerobic cultures. A further increase in glycolytic flux was achieved by simultaneous overexpression of E. coli phosphofructokinase (Pfk) and Pyk with specific glucose consumption and ethanol production rates of 25% and 35% greater, respectively, than the control. Fluxes to lactate were not significantly affected, contrary to previous observations with resting cells harvested from anaerobically growing cultures. To correlate the physiology of resting cells with the physiology of cells prior to harvest, we determined the relevant growth parameters from aerobic and anaerobic precultures. We conclude that glycolytic fluxes in E. coli with submaximal (aerobic) metabolic activity can be increased by overexpression of pyruvate kinases which do not require allosteric activation or co-overexpression with Pfk. However, such improvements require more extensive engineering in E. coli with near maximal (anaerobic) metabolic activity.

Escherichia coli↗
PubMed1992

Homozygous pyruvate kinase deficiency in Hong Kong ethnic minorities.

Three cases of pyruvate kinase (PK) deficiency resulting in congenital haemolytic anaemia with transfusion dependency are described. These cases resulted from consanguineous marriages in non-Han Chinese and include a pair of twins. We believe this to be the first documentation of homozygous PK deficiency in the Hong Kong population. The diagnosis was masked due to transfusion dependency in each case stressing the need to take a sample of pretransfusion blood for PK enzyme assay, and for family studies, when this disorder is suspected.

Anemia, Hemolytic↗
PubMed1987

Regulation of the circadian rhythm of hepatic pyruvate kinase in mice.

We have previously demonstrated circadian variation of pyruvate kinase (PK) activity. PK is a key enzyme of glycolysis, whose activity has been shown to be regulated at the level of enzyme concentration and catalytic activity. To investigate the mechanism of regulation and the components of the circadian rhythm of PK activity, Michaelis-Menten kinetic values were determined. C57BL/6J male mice that had been standardized for over 2 weeks to 12 hr light followed by 12 hr dark (lights on at 0600 hr CST), were killed at 3-hr intervals (six mice/circadian stage) over a 24-hr span. Vmax values, which are a measure of enzyme concentration, were found to be characterized by circadian variation (P = 0.01 by ANOVA, but P = 0.38 by cosinor analysis). The time of highest observed Vmax occurred at the early dark and continued through the entire dark and early light spans. An abrupt decrease in Vmax (-40%) was observed at the end of the light span. PK catalytic activity is regulated by covalent modification (phosphorylation/dephosphorylation) of the enzyme, and the phosphorylated, inactive form of the enzyme has a high Km for its major substrate (1.41 mM phosphoenol pyruvate; PEP). Km values were found to be characterized by a circadian rhythm (P less than 0.001 by cosinor analysis) whose acrophase (1117 hr) was at the beginning of the light cycle (high Km indicates low catalytic activity at subsaturating PEP concentrations). These results indicate that the circadian rhythm for PK activity is composed of at least two components. Enzyme concentration is high during the dark and early light phases but decreases at the end of the light phase. Concentration recovers rapidly within 3 hr after the lights are turned off. Catalytic activity is maximal at a time just after enzyme concentration becomes maximal in the dark phase. Maximal efficiency of the enzyme is thus achieved at the time when animals are eating and glycolysis is operating at high levels.

Animals↗
PubMed1987

Reaction of liver pyruvate kinase with sulfhydryl reagents: effect on its activity.

Homogeneous liver pyruvate kinase was reacted with different sulfhydryl reagents, which included o-iodosobenzoate, 5',5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. Activity determinations of the treated enzyme made with and without Fru(1,6)P2 indicate that the protein contains two sulfhydryl groups per subunit important to its properties, one more accessible than the other. Fru(1,6)P2 added to mixtures prevented loss of activity obtained with o-iodosobenzoate and 5',5'-dithiobis(2-nitrobenzoic acid). It appears that Fru(1,6)P2 does not interfere with the reaction of the reagent with the sulfhydryl group, but prevents an ensuing conformational change, which leads to changes in the enzyme's properties.

Animals↗
PubMed1979

Chronic myelomonocytic leukemia associated with hereditary pyruvate kinase deficiency and multiple acquired erythrocyte abnormalities.

A congenital erythrocyte pyruvate kinase (PK) deficiency was found in a 72-year old female patient with chronic myelomonocytic leukemia (CMML). Erythrocyte PK deficiency was associated with an increase in the activity of hexokinase, 6-phosphogluconate dehydrogenase and glutathione peroxidase in erythrocytes as well as a decrease in acetylcholinesterase, glutathione reductase and glucosephosphate isomerase activities. The enzymatic abnormalities were accompanied by alterations in hemoglobin and in i antigen content of erythrocyte membrane. In addition, bone marrow ultrastructural studies showed dyshemopoietic changes in all blood cell lines and especially in erythroblasts. The present findings confirm the close relationship between CMML and acquired dyserythropoietic syndromes and constitute a new observation of the infrequent association of hereditary erythrocyte enzymopathies and leukemia. A survey of the literature is presented.

Acetylcholinesterase↗
PubMed1998

Platinum complexes and pyruvate kinase activity.

The interaction of platinum complexes with bovine heart pyruvate kinase (PK) was studied by absorption, CD, fluorescence spectroscopy and enzymic activity test. Our results showed that activity of PK was reduced by cis-DDP and potassium tetrachloroplatinate in a time-and concentration dependent manner. Cis-DDP was less effective than K2PtCl4 in reducing PK activity. The native enzyme showed well defined negative Cotton effect at 222 and 208 nm indicating the presence of alpha-helical and beta structure. Platinum binding lowered the Cotton effect in this region by about 10-20% and 30-50% for the system with cis-DDP and K2PtCl4, respectively. Fluorescence study showed that platinum binding quenched tryptophan fluorescence suggesting that binding occurs at the tryptophan residue or its proximity. PK modifications induced by platinum binding would result in a greater resistance to denaturing agents.

Animals↗
PubMed1995

[Pyruvate kinase (PK) isozyme switching and genetic heterogeneity of PK deficiency].

Pyruvate kinase (PK) is a key glycolytic enzyme and has two structural genes; the L/R-gene encodes the L- and R-type PK, whereas the M-gene encodes the M1- and M2-type isozymes. The isozyme switches from the M2 to the R-type during erythroid differentiation, and recent results showed that the switching was achieved by activation of the R-PK promoter activity and the involvement of erythroid-specific transcription factors has been demonstrated. Glycolysis is a major energy source for red cells, therefore, PK deficiency results in hemolysis. PK deficiency is the most common glycolytic enzyme defect associated with hereditary hemolytic anemia, and inherited in an autosomal recessive manner. To date, 46 gene mutations have been identified, and molecular approach might be helpful for diagnosis of PK deficiency, particularly among transfusion-dependent subjects or infantile cases.

Anemia, Hemolytic↗
PubMed1981

Three pyruvate kinase variants with increased affinity for PEP.

Three variants of pyruvate kinase are described which have marked reduction of activity associated with severe non-spherocytic haemolytic anaemia. Each variant shows a reduced K0.5 PEP (the value of the intercept of the abscissa on the Hill plot) and reduced Hill coefficient; FDP activation and ATP inhibition are less than normal and utilization of GDP is increased. The variants are slightly less inhibited by 2,3DPG than controls but require more FDP to relieve this inhibition. Cases 1 and 2 have decreased thermostability but case 3 is normal. The mutant enzymes are further distinguished by their affinity for FDP. Their kinetic and physicochemical properties are compared with other known cases with high affinity for PEP and discussed in terms of a R in equilibrium to T model model for allosteric enzymes.

Adenosine Triphosphate↗
PubMed1978

Isoenzyme of pyruvate kinase in proplastids from developing castor bean endosperm.

Proplastids from developing castor bean (Ricinus communis) endosperm have a pyruvate kinase activity which is extremely unstable on isolation from the organelle. It can be stabilized by 20 mm 2-mercaptoethanol in 20% ethylene glycol. In contrast the soluble pyruvate kinase is stable at 60 C for 10 minutes. The two activities have different pH optima. The soluble and the proplastid activities are eluted from a diethylaminoethyl-Sephadex A-25 sievorptive column at different ionic strengths.

Journal Article↗
PubMed1998

Amino-acid composition of pyruvate kinase M2 isoenzyme variants from rat liver and Morris hepatoma 7777.

Cytosolic fractions B (salted out between 51-70% ammonium sulphate saturation) from rat liver and Morris hepatoma 7777, containing pyruvate kinase (EC 2.7.1.40) M2 isoenzymes, were purified by affinity chromatography on Blue Sepharose CL-6B. When compared by polyacrylamide gel electrophoresis at pH 8.3, all three M2 pyruvate kinase variants from Morris hepatoma 7777 had lower mobilities (alpha2, beta2, gamma3) than the three corresponding variants (alpha1, beta1, gamma2) from normal rat liver. Using an automatic amino-acid analyser, significant differences in selected amino-acid content have been found in corresponding highly purified gamma3 and gamma2 variants from Morris hepatoma and normal rat liver, respectively. The gamma3-variant of the Morris hepatoma M2 isoenzyme had twice the amount of L-tyrosine and L-cysteine, and a content of L-serine higher by 20% than the corresponding gamma2 variant of the normal rat liver M2 isoenzyme. It contained, however, significantly less dicarboxylic amino acids which explains its lower electrophoretic mobility. It showed also a decrease (by about 10%) in several other amino-acid content, corresponding to a 10% decrease in the tumour enzyme molecular mass.

Amino Acids↗