Creatine kinase increases the solubility and enzymatic activity of pyruvate kinase by means of diazymatic coupling

ABSTRACTAllosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

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Estimate of the Intrafamilial Correlation for Serum Creatine Kinase and Pyruvate Kinase in Females at Risk for Duchenne and Becker Muscular Dystrophies

Human Heredity ◽

10.1159/000154029 ◽

1991 ◽

Vol 41 (6) ◽

pp. 370-378

Author(s):

Eliete Rabbi-Bortolini ◽

Gloria Maria D.Dal Colletto ◽

Maria Rita Passos-Bueno ◽

Mariz Vainzof ◽

Debora Rapaport ◽

...

Keyword(s):

At Risk ◽

Creatine Kinase ◽

Pyruvate Kinase ◽

Serum Creatine Kinase ◽

Muscular Dystrophies

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Detection of carriers of X-linked gene for Duchenne muscular dystrophy by levels of creatine kinase and pyruvate kinase

Journal of the Neurological Sciences ◽

10.1016/0022-510x(83)90197-1 ◽

1983 ◽

Vol 62 (1-3) ◽

pp. 171-180 ◽

Cited By ~ 1

Author(s):

Daisy Neves Falcão-Conceição ◽

Marcia Mattos Gonçalves-Pimentel ◽

Marcia Leite Baptista ◽

Sônia Ubatuba

Keyword(s):

Creatine Kinase ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Pyruvate Kinase ◽

Linked Gene

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Creatine measurement in serum and urine with an automated enzymatic method

Clinical Chemistry ◽

10.1093/clinchem/39.8.1613 ◽

1993 ◽

Vol 39 (8) ◽

pp. 1613-1619 ◽

Cited By ~ 10

Author(s):

C Beyer

Keyword(s):

Creatine Kinase ◽

Lactate Dehydrogenase ◽

Pyruvate Kinase ◽

Present Method ◽

Comparative Method ◽

Reference Interval ◽

Enzymatic Method ◽

Men And Women ◽

Enzymatic Procedure ◽

Serum And Urine

Abstract I describe an automated enzymatic procedure to quantitate creatine in both serum and urine. In this assay, which requires no pretreatment of the sample, creatine kinase (CK; EC 2.7.3.2) and pyruvate kinase (EC 2.1.7.40) are used as auxiliary enzymes and lactate dehydrogenase (EC 1.1.1.27) is used in the indicator reaction. CK is also used as the starting reagent. Data obtained with the present method for creatine measurement in serum were compared with those from the Jaffé method and an enzymatic method: y = 1.13x - 7.58, SE = 4.48, and r = 0.925 (Jaffé); and y = 1.17x + 2.73, SE = 5.06, and r = 0.962 (enzymatic); for creatine measurement in urine: y = 0.63x + 39.74, SE = 296.7 and r = 0.719 (Jaffé). The present method provides improved precision: the total CVs for serum, determined by the present and comparative methods, respectively, were 3.5-8.9%, 8.2-43.0%, and 5.3-16%; for urine, the CVs were 3.3-5.1% and 9.6-21.2% for the present and comparative method, respectively. I established the normal reference interval as 13-74 and 13-89 mumol/L for creatine in serum, and as 175-700 and 150-1200 mumol/24 h for creatine in urine for men and women, respectively.

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Contraction-induced injury to the extensor digitorum longus muscles of rats: the role of vitamin E

Journal of Applied Physiology ◽

10.1152/jappl.1997.83.3.817 ◽

1997 ◽

Vol 83 (3) ◽

pp. 817-823 ◽

Cited By ~ 34

Author(s):

Jack H. Van Der Meulen ◽

Anne McArdle ◽

Malcolm J. Jackson ◽

John A. Faulkner

Keyword(s):

Creatine Kinase ◽

Vitamin E ◽

Pyruvate Kinase ◽

Extensor Digitorum Longus ◽

Muscle Fibers ◽

Extensor Digitorum ◽

Intravenous Injections ◽

Antioxidant Function ◽

The Difference

Van der Meulen, Jack H., Anne McArdle, Malcolm J. Jackson, and John A. Faulkner. Contraction-induced injury to the extensor digitorum longus muscles of rats: the role of vitamin E. J. Appl. Physiol. 83(3): 817–823, 1997.—Three days after a protocol of 225 pliometric (lengthening) contractions was administered to in situ extensor digitorum longus muscles of rats, the force deficit was 64 ± 7% and the percentage of damaged muscle fibers was 38 ± 5% of the control values. We then tested the hypothesis that at 3 h and 3 days after the protocol an elevation in the muscle vitamin E content would decrease the force deficit, the percentage of damaged muscle fibers, and the serum activities of creatine kinase and pyruvate kinase. The 5–8 days of intravenous injections of α-tocopherol increased muscle vitamin E content threefold compared with vehicle (ethanol)-treated rats. Despite the difference in vitamin E content, the force deficit and number of damaged fibers were not different. After the contraction protocol, the serum creatine kinase and pyruvate kinase activities of the vehicle-treated rats increased fourfold at 3 h and twofold at 3 days, whereas the vitamin E-treated rats showed no change. We conclude that vitamin E treatment did not ameliorate either the induction of the injury or the more severe secondary injury at 3 days. Despite the absence of evidence for an antioxidant function, the lack of any increase in serum enzyme activities for vitamin E-treated rats at 3 h and 3 days supported a role for vitamin E in the prevention of enzyme loss after muscle damage.

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

Biochemical Journal ◽

10.1042/bj1350265 ◽

1973 ◽

Vol 135 (2) ◽

pp. 265-276 ◽

Cited By ~ 19

Author(s):

B. Focant ◽

D. C. Watts

Keyword(s):

Creatine Kinase ◽

Smooth Muscle ◽

Pyruvate Kinase ◽

Conformational Changes ◽

Intermediate Stage ◽

Hill Coefficient ◽

Rabbit Muscle ◽

Difference Spectra ◽

Muscle Creatine Kinase ◽

Quaternary Complex

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 NO3- or Cl- ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by αβ-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 NO3- ions to the enzyme–creatine–MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme–creatine–NO3-–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 NO3- is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme–pyruvate–NO3-–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 α-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.

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Creatine-kinase (CK) and pyruvate-kinase (PK) activities in cord blood of normal newborn infants: Application to duchenne muscular dystrophy screening programs

American Journal of Medical Genetics ◽

10.1002/ajmg.1320160308 ◽

1983 ◽

Vol 16 (3) ◽

pp. 367-372 ◽

Cited By ~ 3

Author(s):

Maria Rita Passos ◽

Mayana Zatz

Keyword(s):

Creatine Kinase ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Cord Blood ◽

Pyruvate Kinase ◽

Newborn Infants ◽

Screening Programs

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Serum creatine kinase and pyruvate kinase activities in normal adolescent females

Journal of the Neurological Sciences ◽

10.1016/0022-510x(82)90198-8 ◽

1982 ◽

Vol 54 (3) ◽

pp. 349-352 ◽

Cited By ~ 1

Author(s):

I.R. Livingstone ◽

D. Gardner-Medwin ◽

R.J.T. Pennington ◽

J.N. Walton

Keyword(s):

Creatine Kinase ◽

Pyruvate Kinase ◽

Adolescent Females ◽

Serum Creatine Kinase

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Co-localization and functional coupling of creatine kinase B and gastric H+/K+-ATPase on the apical membrane and the tubulovesicular system of parietal cells

Biochemical Journal ◽

10.1042/bj3110445 ◽

1995 ◽

Vol 311 (2) ◽

pp. 445-451 ◽

Cited By ~ 18

Author(s):

E A Sistermans ◽

C H W Klaassen ◽

W Peters ◽

H G P Swarts ◽

P H K Jap ◽

...

Keyword(s):

Creatine Kinase ◽

Atpase Activity ◽

Pyruvate Kinase ◽

Apical Membrane ◽

Immunogold Labelling ◽

Parietal Cells ◽

Functional Coupling ◽

Experimental Conditions ◽

Atp Levels ◽

Creatine Kinase B

Immunogold labelling of creatine kinase B (BB-CK) and gastric H+/K(+)-ATPase in the parietal cells of the stomach revealed colocalization of these two enzymes on the apical membrane and the membranes of the tubulovesicular system. Upon fractionation of hog parietal cells, a specific fraction of the BB-CK proteins remained associated with the purified vesicles, in which gastric H+/K(+)-ATPase is highly enriched. The BB-CK present in this highly purified preparation was able to support pronounced H+/K(+)-ATPase activity in K(+)-loaded vesicles in the presence of phosphocreatine and ADP, although only low levels of ATP were measured. In contrast, when pyruvate kinase, phosphoenolpyruvate and ADP were used as an ATP-generating system to sustain similar levels of H+/K(+)-ATPase activity, ATP levels were more than 10-fold higher. Changing the experimental conditions such that ATP levels were the same for both systems resulted in significantly elevated H+/K(+)-ATPase activities in the BB-CK/phosphocreatine system in comparison with the pyruvate kinase/phosphoenolpyruvate system. These results indicate that gastric H+/K(+)-ATPase has preferential access to ATP generated by creatine kinase co-localized on the membranes of the vesicles.

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