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PubMed1991

Positive and negative regulation of gene expression by insulin and glucagon: the model of L-type pyruvate kinase gene.

L-type pyruvate kinase gene regulation is an excellent model of gene control by hormones and diet. In vivo and ex vivo experiments allowed us to established that thyroid hormones and glucocorticoids act on pyruvate kinase gene expression at the post-transcriptional level. In contrast, glucose and insulin together stimulate transcription of this gene while glucagon inhibits it. Insulin or glucose are individually inefficient and glucagon-dependent transcriptional inhibition seems to be dominant in insulin + glucose-dependent activation. A 14-kbp fragment encompassing the entire pyruvate kinase gene and 3.2-kbp of 5' flanking sequences is expressed in transgenic mice exactly like the endogenous gene; the 3.2-kbp upstream region is sufficient to confer this tissue-specific and hormone/diet-regulated expression to reporter genes. In vivo, DNAse I hypersensitivity analysis revealed the presence of 3 liver-specific groups of hypersensitive sites (HSS). The proximal sites, between + 1 and -183 bp with respect to the start site of transcription, were, in addition, transcription-dependent. The nature and functional role of proteins binding to this proximal upstream sequence were analyzed by in vitro binding and cell free transcription experiments. The existence of more upstream cis-acting elements was investigated by transient transfection assays using differentiated hepatoma cell lines and hepatocytes in primary culture. These experiments permitted the detection of an extinguisher active in hepatoma Hep G2 cells but not in hepatocytes, and of an activating element which could correspond to a distal HSS. Unfortunately, this investigation has not yet allowed us to determine with accuracy the DNA elements responsible for response to diet and hormones.

Animals↗
PubMed1997

Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase.

Properties of mung bean pyruvate kinase were studied and the active site groups were derived. Metabolites like AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate, 3-phospho-glycerate, isocitrate, malate and alpha-ketoglutarate had practically no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. Some metabolites such as ATP, GTP, and UMP were found to be weakly inhibitory. Moderate to strong inhibition was observed with citrate, succinate, glutamate and oxalate. Inhibition brought about by ATP and citrate when present together showed synergistic effect. Inhibition by citrate was non-competitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2+. Small concentrations of the SH specific reagents, namely iodoacetamide (0.1 and 0.2 mM), N-ethylmaleimide(0.05-0.1 mM) and p-chloromercuribenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in the presence of methylene blue (100 and 200 micrograms/ml) and rose bengal (5 and 10 micrograms/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate (DEP), a time dependent exponential decay in its activity was observed with a parallel increase in absorbance at 240 nm. PEP protected the enzyme against inactivation by DEP. Reagents specific for tyrosine (iodine and tetranitromethane) and tryptophan residues (N-bromosuccinimide) residues had no effect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.

Binding Sites↗
PubMed1976

The proton transfer reactions catalyzed by yeast pyruvate kinase.

1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton-transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10-100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction of pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase (Robinson, JL. and Rose, I.A. (1972) J. Biol. Chem. 247, 1096-1105). The pH-profiles and the divalent cation dependencies were similar for Fru-1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization by yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization by the muscle enzyme which proceeds when ATP is replaced by Pi or other dianions. 3. Fructose-1,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme.

Adenosine Diphosphate↗
PubMed1972

The role of mitochondria in modifying calcium-sensitive cytoplasmic metabolic activities. Modification of pyruvate kinase activity.

1. The modification of pyruvate kinase activity in vitro was examined by altering the environmental [Mg(2+)]/[Ca(2+)] ratio with EDTA on the one hand and isolated rat liver mitochondria on the other. 2. Controlled additions of Ca(2+) and EDTA caused pyruvate kinase activity to be alternately and rapidly switched on and off. 3. By being able to accumulate Ca(2+) in preference to Mg(2+) rat liver mitochondria were able to alter the [Mg(2+)]/[Ca(2+)] ratio in the vicinity of pyruvate kinase and thereby modify the activity of this enzyme. 4. The possible role of mitochondria in modifying pyruvate kinase and other ion-sensitive cytoplasmic enzyme activities is discussed.

Animals↗
PubMed1993

Water-induced transitions in the K+ requirements for the activity of pyruvate kinase entrapped in reverse micelles.

The activity of pyruvate kinase was studied in reverse micelles formed with cetyltrimethylammonium bromide, n-octane, hexanol, and various amounts of water. In systems with 100% water, K+ is an essential activator of pyruvate kinase [Kachmar, J. F., & Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683]; i.e., without and with K+, the activities observed were 0.07 and 300 mumol/(min.mg), respectively. In the micellar system with 3.6% water (v/v), pyruvate kinase exhibited an activity of about 45 mumol/(min.mg), in the absence of K+. The kcat was about 450 times larger than that in 100% water without K+. Km values for ADP and phosphoenolpyruvate differed, but not markedly from those in 100% water with or without K+. The kinetics of pyruvate kinase in reverse micelles were not affected by K+. The activity curve of pyruvate kinase in reverse micelles without K+ in a pH range of 6.0-8.5 was almost superimposable to that of the enzyme in 100% water with K+, and it differed drastically from that in 100% water without K+. The fluorescence emission spectra of pyruvate kinase in 100% water exhibited a blue shift of 3 nm upon the addition of ligands (Mg2+, phosphoenolpyruvate, and K+) that cause a transition of the enzyme to its active state. Without ligands, the entrapment of pyruvate kinase in reverse micelles with 3.0% water produced a blue shift of nearly 2 nm with respect to that of the enzyme in 100% water without ligands. As water was raised to 7.0% (v/v), the maximal emission shifted to longer wavelengths; these changes paralleled the appearance of the K(+)-dependent activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Crystallization and preliminary analysis of enzyme-substrate complexes of pyruvate kinase from rabbit muscle.

Pyruvate kinase from rabbit muscle has been crystallized in a form suitable for high resolution X-ray analysis. Complexes of the enzyme with Mn2+ and either pyruvate or oxalate crystallize from solutions of polyethyl-eneglycol 8000 at pH 6.0. Crystals obtained from solutions of the complexes with pyruvate or oxalate appear isomorphous and belong to the triclinic space group P1. The crystals have unit cell dimensions a = 83.3(4) A, b = 109.4(6) A, c = 145.7 (7) A, alpha = 94.9 degrees, beta = 93.6 degrees, gamma = 112.2 degrees. These crystals diffract to better than 2.4 A resolution and are stable in the X-ray beam for at least 20 hr. Electron paramagnetic resonance measurements on a single crystal show that Mn2+ is bound to the crystalline protein.

Animals↗
PubMed1976

L-type pyruvate kinase from human liver. Purification by double affinity elution, electrofocusing and immunological studies.

L-type pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was highly purified from adult human liver. This purification included ammonium sulphate fractionation, DEAE-Sephadex batchwise absorption and two CM-Sephadex chromatographies with selective elution by ligands; in the former chromatography pyruvate kinase was eluted by ATP, in the latter one by phosphoenolpyruvate and fructose 1,6-diphosphate. The last step of the purification procedure involved a hydroxyapatite column chromatography. This purification procedure allowed us to obtain 3.6 mg of protein with a specific activity 190 I.U./mg, i.e. a 1200-fold purification with an overall yield of about 8%. This preparation was homogenous as judged by immunodiffusion, acrylamide and sodium dodecyl sulphate acrylamide gel electrophoresis. Anti L-type pyruvate kinase antibodies were obtained from rabbits and the antigenic properties of L-type pyruvate kinase were studied. The enzyme appeared to be a tetramer (molecular weight 220 000-240 000) with subunits of similar molecular weight about 60 000). Two interconvertible major forms were found by isoelectrofocusing in a sucrose gradient and in an acrylamide slab gel: one had an isoelectric point of 5.85 +/- 0.09 and was the major enzymatic form after incubation with fructose 1,6-diphosphate or high concentrations or SH reagents. The other form (isoelectric point 6.28 +/- 0.03) was the major form of L-type pyruvate kinase in liver crude extract, and after incubation of purified enzyme with a proteic fraction isolated from liver extract by ammonium sulphate precipitation.

Chromatography, Affinity↗
PubMed2002

Pyruvate kinase from Chlamydia trachomatis is activated by fructose-2,6-bisphosphate.

Pyruvate kinase is the final regulatory point in the catabolic Embden-Meyerhoff-Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacterium Chlamydia trachomatis and demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purified C. trachomatis pyruvate kinase. The results indicate that C. trachomatis pyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show that C. trachomatis pyruvate kinase requires both K+ and Mg2+ ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis-Menten kinetics with respect to ADP. In addition, C. trachomatis pyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibits C. trachomatis pyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase, C. trachomatis pyruvate kinase was allosterically activated by fructose-2,6-bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.

Allosteric Regulation↗
PubMed1979

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.

Animals↗
PubMed1983

The effect of dexamethasone on pyruvate kinase activity in primary cultures of hepatocytes.

Pyruvate kinase activity in primary cultures of hepatocytes isolated from a normal rat was maintained at a constant level similar to that found in vivo (14.0 +/- 2.8 units per mg of DNA) for over 6 days when both dexamethasone and insulin were included in the medium. Yet the pyruvate kinase activity decreased 50% when the cells were cultured for 2 days and 4 days, respectively, in the presence of either dexamethasone or insulin alone. A brief, 10 min incubation of hepatocytes in the presence of dexamethasone was sufficient to maintain the enzyme activity of cells subsequently cultured for 4 days in the presence of insulin. The optimal dexamethasone concentration was 1 microM. Three other glucocorticoids were able to maintain the pyruvate kinase activity in cells cultured in medium containing insulin. The presence of the protein synthesis inhibitors, actinomycin D or cyclohexamide in cells cultured in the presence of dexamethasone and insulin resulted in a 25% decrease in the pyruvate kinase activity. Therefore, it is suggested that the synergistic effect of glucocorticoids and insulin to maintain pyruvate kinase activity in primary cultures of hepatocytes is dependent upon the ability of these cells to maintain protein synthesis.

Animals↗
PubMed1982

A kinetic analysis of the distinct regulatory sites on rabbit muscle pyruvate kinase.

Interaction of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with inhibitory and activatory effectors was analyzed in terms of the Yagi-Ozawa equation. Straight-line plots obtained for L-phenylalanine + L-alanine and L-phenylalanine + L-phenylalanyl methyl ester (PheAlaOMe) indicated competition for common sites. Parabolic plots for 2-phosphoglycerate and L-phenylalanine or PheAlaOMe suggested lack of competition between these compounds. Biphasic concave plots were obtained for 2-phosphoglycerate and L-1-amino-2-phenylethyl phosphonic acid (PnPhe), and mixtures of inhibitory (L-phenylalanine, PheAlaOMe) and activatory (PnPhe, L-alanine and L-1-aminoethyl phosphonic acid (PnAla)) amino acid derivatives. The latter non-classical Yagi-Ozawa plots can be interpreted in terms of the existence of distinct regulatory sites for inhibitory and activatory ligands.

Alanine↗
PubMed2003

A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds.

Cytosolic pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is an important glycolytic enzyme, but the post-translational regulation of this enzyme is poorly understood. Sequence analysis of the soybean seed enzyme suggested the potential for two phosphorylation sites: site-1 (FVRKGS220DLVN) and site-2 (VLTRGGS407TAKL). Sequence- and phosphorylation state-specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc) is phosphorylated at both sites in vivo. However, by SDS-PAGE, the phosphorylated polypeptides were found to be smaller (20-51 kDa) than the full length (55 kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in developing seeds was determined to be of approximately 1900 kDa. In vitro, the 26S proteasome degraded associated PyrKinc polypeptides, and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some part of the phosphorylated PyrKinc was conjugated to ubiquitin and shifted to high molecular mass forms in vivo. Moreover, recombinant wild-type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated polypeptide of approximately 51 kDa was observed that was in parallel with a loss of the full-length 55 kDa polypeptide. Interestingly, the C-terminal 51 kDa truncation showed not only pyruvate kinase activity but also activation by aspartate. Collectively, the results suggest that there are two pathways for PyrKinc modification at the post-translational level. One involves partial C-terminal truncation to generate a 51 kDa pyruvate kinase subunit which might have altered regulatory properties and the other involves phosphorylation and ubiquitin conjugation that targets the protein to the 26S proteasome for complete degradation.

Amino Acid Sequence↗
PubMed1982

Long-term modulation of type L pyruvate kinase activity in young and mature rats.

The regulation of type L pyruvate kinase concentrations in liver of young (35-45 days old) and adult (60-85 days old) rats starved and re-fed a 71% sucrose diet was investigated. Re-feeding is accompanied by an increase in the enzyme level in liver determined kinetically and immunologically. A constant ratio of kinetic activity to immunological activity was observed under all conditions examined, indicating that activity changes are the result of a regulation of synthesis or degradation and not an interconversion between kinetically active and inactive forms of the enzyme. Synthesis of pyruvate kinase was directly examined by using hepatocytes isolated from starved and re-fed rats. A stimulation of pyruvate kinase synthesis is observed on re-feeding. This increase in synthesis of pyruvate kinase is retained by the isolated hepatocyte for up to 7h in the absence of hormonal stimuli. Administration of glucagon (1mum) to the isolated hepatocytes had no influence on synthesis of pyruvate kinase and no evidence for a glucagon-directed degradation of the enzyme was found. Re-feeding the rat was followed by a transient increase in the synthesis of pyruvate kinase. The peak rate of synthesis was observed before a detectable increase in the enzyme concentration. After a rapid synthesis period, a new steady-state level of the enzyme was achieved and synthesis rates declined. The time course and magnitude for the response to the sucrose diet was dependent on the age of the rat. In young rats, an increase in pyruvate kinase synthesis is observed within 6h and peak synthesis occurs at 11h after re-feeding sucrose. The peak synthesis rate for pyruvate kinase for young rats represents approx. 1% of total protein synthesis. With adult rats, increased pyruvate kinase synthesis is not observed for 11h, with peak synthesis occurring at 24h after re-feeding. In the older rats, peak pyruvate kinase synthesis constitutes greater than 4% of total protein synthesis. Continued re-feeding of the adult rat beyond 24h is accompanied by a decline of pyruvate kinase synthesis to approx. 1.5% of total protein synthesis. The concentration of the enzyme, however, does not decline during this period, suggesting that control of pyruvate kinase degradation as well as synthesis occurs.

Age Factors↗
PubMed1981

Pyruvate kinase: activation by and catalytic role of the monovalent and divalent cations.

This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K+ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact, E. coli synthesize, depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4-8 A of the divalent cation activator and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg+2 is optimal for the physiological reaction, however, Co+2, Mn+2, and Ni+2 also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.

Animals↗
PubMed1977

Inactivation of pyruvate kinase type M2 from chicken liver by phosphorylation, catalyzed by a cAMP-independent protein kinase.

A cAMP-independent protein kinase from chicken liver phosphorylated and inactivated pyruvate kinase type M2 from the same tissue. Complete inactivation was reached when 4 mol of phosphate were incorporated/mol of tetrameric pyruvate kinase. The protein kinase bound with high affinity to pyruvate kinase type M2 (Km value for pyruvate kinase = 6 X 10(-10)M; it phosphorylated phosvitin and casein but not histones, ATP and GTP were substrates. The differences between the properties of this protein kinase in the interconversion of pyruvate kinase and that described previously are discussed.

Animals↗
PubMed1982

Pyruvate kinase deficiency anaemia in a Basenji dog.

Pyruvate kinase deficiency anaemia was suspected in an 18-month-old male Basenji dog after other known causes of canine haemolytic anaemia had been excluded. Anaemia of moderate severity (packed cell volumes 0.20 to 0.26 1/1) and reticulocytosis (uncorrected reticulocyte counts 8 to 43%) persisted during 5 months' observation, and biopsies showed development of bone marrow fibrosis and sclerosis. The diagnosis of pyruvate kinase deficiency anaemia was presumptive because erythrocyte pyruvate kinase concentrations in the affected dog were inconclusive and related animals were not available for enzyme assay. However, the gene for pyruvate kinase deficiency is known to occur among Basenji dogs in Australia.

Anemia, Hemolytic, Congenital↗
PubMed2005

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum.

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K(m) of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1mM pyridoxal-5'-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K(i) of 0.8mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.

Amino Acid Sequence↗
PubMed1973

Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase.

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO(3) (-) or Cl(-) ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by alphabeta-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO(3) (-) ions to the enzyme-creatine-MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme-creatine-NO(3) (-)-MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO(3) (-) is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme-pyruvate-NO(3) (-)-MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or alpha-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K(+)-free enzyme. Only the former inhibits the K(+)-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.

Adenosine Diphosphate↗