Stereochemistry of pyruvate kinase, pyruvate carboxylase, and malate enzyme reactions.
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Chemical Phenomena↗
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Use of serum creatine kinase, pyruvate kinase, and genetic linkage for carrier detection in Duchenne and Becker dystrophy.
Carrier detection in Duchenne dystrophy (DD) and Becker dystrophy (BD) can be achieved with DNA probes that recognize restriction fragment length polymorphisms (RFLPs). In 22 families, we found that 16 of 23 females at risk for being DD or BD carriers could be provided with more definitive indications of carrier status beyond the use of creatine kinase/pyruvate kinase and pedigree analysis. RFLP analysis was not possible for six individuals despite potentially informative probes, because family members critical to the analysis were unavailable. In only one instance were all eight probes uninformative.
Creatine Kinase↗
Normalisation of red blood cell pyruvate kinase in pyruvate kinase deficiency by riboflavin treatment.
A patient with an erythrocyte glutathione reductase activity of 50% of the normal value and an abnormal pyruvate kinase (PK) was given 36 mg riboflavin daily for 6 months. The glutathione reductase activity was restored and the abnormal pyruvate kinase was converted to normal. The clinical state of the patient improved. It can be concluded that the abnormality of PK, at least with this patient, is a secondary effect. Therefore, it is suggested that other abnormalities be searched for when an altered PK is detected. Treatment of this abnormality will help the patients more efficiently.
Carbohydrate Metabolism, Inborn Errors↗
Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L from Rana pipiens.
The regulatory properties of type L pyruvate kinase from Rana 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: i.e. 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.
Animals↗
Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase.
Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.
Animals↗
Regulation of glycolysis in Neurospora crassa. Kinetic properties of pyruvate kinase.
Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.
Adenosine Triphosphate↗
[Sensitivity of fructose diphosphate binding sites of yeast pyruvate kinase to pyruvate kinase antibodies].
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Antibodies↗
Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: pyruvate kinase.
Pyruvate kinase (EC 2.7.1.40) from Azotobacter vinelandii responds sharply to the adenylate energy charge, with a decrease in activity at high values of charge, as expected for an enzyme of an adenosine triphosphate-regenerating sequence. Glycolytic intermediates, especially glucose 6-phosphate, fructose 6-phosphate, and fructose-1,6-diphosphate, strongly stimulate the reaction and overcome the inhibition caused by high values of energy charge. Thus, the properties of this enzyme depend on interaction between energy charge and the concentrations of hexose phosphates. The properties of pyruvate kinase, together with those of phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase, seem adapted to provide appropriate partitioning of phosphoenolpyruvate between competing pathways in response to metabolic need.
Adenine Nucleotides↗
Calcium-like action of phenethylbiguanide and related compounds: inhibition of pyruvate kinase.
Pyruvate kinase (EC 2.7.1.40) is inhibited by phenethylbiguanide. The kinetics of inhibition are competitive between biguanide and divalent, but not monovalent, metal cation activators of the enzyme; biguanide inhibition thus resembles inhibition by Ca(++). Alteration of either the polar or nonpolar portion of the phenethylbiguanide molecule quantitatively reduces its effectiveness as an inhibitor of pyruvate kinase, but the kinetics of inhibition remain qualitatively unchanged. Measurements of [(3)H]phenethylbiguanide binding to the enzyme indicate the presence of a single class of about 12 binding sites per enzyme molecule; binding characteristics are not significantly different in the presence of either monovalent or divalent metal cations. Studies with (45)Ca(++) and (54)Mn(++) demonstrate about 4 metal binding sites per enzyme molecule; phenethylbiguanide displaces these metal cations from the enzyme. Studies with several enzymes, dependent upon divalent metal cations, of both metal-bridge and substrate-bridge classes fail to show significant inhibition except at much higher phenethylbiguanide concentrations.
Animals↗
A monomeric form of pyruvate kinase in human pyruvate kinase deficiency.
A mutant pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human erythrocytes which is easily separated into monomers and dimers by gel chromatography is described. Tht mutant enzyme shows almost the same pH optimum and thermostability as normal enzyme, but has a decreased stability on shaking with air, a decreased Km for phosphoenolpyruvate and a loss of allosteric properties. The apparent Km values for phosphoenolpyruvate of tetramers and monomers were the same. The tetrameric enzyme was slightly activated by fructose-1,6-diphosphate but the monomeric form was not. The tetrameric enzyme was found to dissociate spontaneously to dimeric and monomeric forms.
Adenosine Diphosphate↗
Insulin activates 6-phosphofructo-2-kinase and pyruvate kinase in the liver. Indirect evidence for an action via a phosphatase.
The effect of insulin on hepatic glucose production has been studied in anesthetized rats in the postabsorptive state. Insulin decreases significantly hepatic glucose production within 5-10 min. It also increases the level of fructose 2,6-bisphosphate, via an increase in the Vmax of 6-phosphofructo-2-kinase and concomitantly decreased the activity of fructose-2,6-bisphosphatase, resulting in a 5-fold increase in the ratio of kinase/phosphatase. Insulin also increased the apparent Kd of pyruvate kinase for phosphoenolpyruvate. The changes in the activity of 6-phosphofructo-2-kinase and pyruvate kinase were measured after separation from possible modulators, and suggest a decrease in their phosphorylation state which cannot be attributed to a decrease in the level of cAMP or in the activity of cAMP-dependent protein kinase since these two parameters were not modified by insulin. In addition, neither the activity of phosphorylase a nor that of glycogen synthase were modified. The data strongly suggest that the increase in the glycolytic rate plays a role in the effect of insulin on hepatic glucose production and that insulin mediates its effect on the activity of these enzymes via one or more phosphatases.
Adenosine Triphosphate↗
Effect of desdanine on nucleoside diphosphate kinase and pyruvate kinase of Escherichia coli.
Kinetic analysis demonstrated that the irreversible inhibition of nucleoside diphosphate kinase of Escherichia coli by desdanine proceeds via a reversible Enzyme-Inhibitor complex. It is known that pyruvate kinase of E. coli becomes inactive upon prolonged dialysis in the absence of a reducing reagent, such as dithiothreitol and that the inactive enzyme is reactivated if dithiothretiol is added. Desdanine inhibits this reactivation process. The effect is discussed in relation to inhibition in relation to the inhibition of growth of E. coli by desdanine under anaerobic conditions. Pyruvate kinase of E. coli changes not only in intracellular quantity but in its kinetic characteristics depending on growth conditions, aerobic or anaerobic. The enzyme shows a broad specificity for nucleside diphosphates, especially in the presence of AMP, and thus resembles closely nucleoside diphosphate kinase. The in vivo role of pyruvate kinase in supplying nucleoside triphosphates under anaerobic conditions is discussed.
Acrylamides↗
Serum creatine kinase and pyruvate kinase in Duchenne muscular dystrophy carrier detection.
The incidence of elevated serum creatine kinase (CK) and pyruvate kinase (PK) activities was compared in 20 definite carriers of Duchenne muscular dystrophy (DMD), 47 possible carriers, and 42 female controls. When adult age was not regarded as a variable, 70% of the definite carriers had elevated PK, 55% had elevated CK, and 75% had elevated PK or elevated CK or both, 38% of the possible carriers had elevated PK, 19% had elevated CK, and 40% had elevated PK or elevated CK or both. The detection efficiency of the CK test was influenced by the age of the subjects: the upper normal limit of serum CK in the adult controls was at the minimum between 21 and 35 years of age, and CK activity in some carriers declined from elevated to normal levels with increasing age. With these considerations, 70% of definite carriers had elevated CK and 80% had elevated PK and/or CK; 34% of the possible carriers had elevated CK and 43% had elevated PK and/or CK. On the basis of the PK and CK measurements, only 16 of 24 possible carrier mothers were likely to be DMD carriers, implying that the other 8 were non-carrier mothers of new mutant sons.
Adolescent↗
Estimate of the intrafamilial correlation for serum creatine kinase and pyruvate kinase in females at risk for Duchenne and Becker muscular dystrophies.
In order to verify the possibility of nonrandom X-inactivation in females heterozygous for Duchenne (DMD) and Becker (BMD) muscular dystrophies, intrafamilial correlations and the heritabilities for serum creatine kinase (CK) and pyruvate kinase (PK) were estimated in a large sample of females belonging to families with affected patients. The results of the present investigation suggest that the apparent intrafamilial correlations for serum CK reported in previous studies in DMD families are not related with the presence of the DMD/BMD gene. Our data do not seem to support the hypothesis of a gene leading to a preferential inactivation of the X-chromosome in females at risk for the dystrophin gene.
Adolescent↗
Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase.
We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
Acetate Kinase↗
Clinical investigation in Duchenne dystrophy: V. Use of creatine kinase and pyruvate kinase in carrier detection.
We have determined the value of creatine and pyruvate kinase (CK and PK) in carrier detection by evaluating 811 females in 73 families participating in the Collaborative Investigation of Duchenne Dystrophy. Thirty-nine obligate carriers, 244 normal controls (paternal females), as well as 76 possible carriers and 351 carrier suspects had three CK and PK specimens analyzed at a central laboratory. The CK and PK values varied with age in normals: both fell with age early in life, and CK rose after the fifth decade. Discriminant analysis indicated that the combination of mean CK and PK corrected for age yielded the best data for calculation of carrier probability. Using the best model, only 45% of obligate carriers could be identified at a false-positive rate of 2.5%. Daughters of obligate carriers have a disproportionate decline in CK and PK with age as compared to noncarrier females, suggesting that the rate of carrier detection will be higher in the first two decades. Our low rate of carrier detection, as compared to other studies, may reflect both the age of our obligate carrier population and the use of a control group that is more representative of the carrier population.
Adolescent↗
Adrenergic control of the cyclic AMP-dependent protein kinase and pyruvate kinase in isolated hepatocytes. Application of a synthetic peptide substrate for measuring protein kinase activity.
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Adrenergic alpha-Agonists↗
Studies on the cyclic 3':5'-AMP-stimulated pig liver protein kinase reaction with pyruvate kinase as substrate.
The phosphorylation of pig liver pyruvate kinase by cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been studied. For comparison, mixed histone and a synthetic heptapeptide were also used as substrates. Protein kinase was purified by chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was stimulated by cyclic AMP with apparent Ka values of 2.5 and 0.8 x 10-7 M for pyruvate kinase and histone substrates, respectively. Divalent cations were essential for the activity of the protein kinase. Variation of the concentration of ATP resulted in approximately straight lines in Lineweaver-Burk plots for the phosphorylation of both pyruvate kinase and mixed histone. The apparent Km values for ATP were 21 and 11 muM, respectively. The phosphorylation rate increased with the concentration of pyruvate kinase even at a concentration of 2 muM pyruvate kinase. At a high ionic strength, the phosphorylation rate of both pyruvate kinase and histone decreased. The phosphorylation rate varied markedly with pH in imidazole/HC1 and Tris/HC1 buffers. At slightly alkaline pH values, pyruvate kinase was phosphorylated at a much higher rate than pH7, but this was not the case for histone. At pH 8.5, the phosphorylation rate of pyruvate kinase was 3.5 times the rate at pH 7, while the corresponding increase for the histone phosphorylation was 50 per cent. In potassium phosphate buffers, the phosphorylation rate of both substrates did not change significantly over the pH range studied. Arrhenius' plots of the protein kinase reaction resulted in a break at about 10 degrees when pyruvate kinase was used as substrate, whereas a straight line was obtained when using histone. The negative allosteric effectors of pyruvate kinase, alanine, and phenylalanine, increased the phosphorylation rate of pyruvate kinase at pH 8 by 50 and 120 per cent, respectively. The same effectors did not influence the phosphorylation rate of mixed histone or a synthetic heptapeptide. It is concluded that the conformations adopted by pyruvate kinase in the presence of allosteric inhibitors make it a better substrate for the protein kinase.
Adenosine Triphosphate↗