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

Five separate methods, paper chromatography, ethanol solubility, pH dependence, enzymatic activity, and NMR saturation transfer, were used to study the coupling of pyruvate kinase (PK) and creatine kinase (CK). Each method demonstrated specific coupling of the two proteins. The coupling with CK showed that PK had increased ethanol solubility, enzymatic activity, pH-dependent aqueous solubility, and ability to receive ATP directly from CK even at very high (1.2 M) ionic strength at equilibrium conditions. The robustness of the coupling between two enzymes with highin vivo concentrations indicates the necessity for considering kinetic schemes utilizing high enzyme concentrations, as opposed to the dilute properties of Michaelis — Menten kinetics. The direct transfer of ATP from PK to CK constitutes a net transfer of the glycolytically produced high-energy phosphate onto creatine, maintaining a high ADP and low ATP concentration in the vicinity of the glycolytic complex, conditions suited to maintaining a high glycolytic flux. The demonstration of the physical and functional coupling of two soluble enzymes, termed diazymes, suggests that glycolysis may be regulated by diazymatic coupling and that other cellular processes could be regulated similarly.     

Creatine kinase in regulation of heart function and metabolism. I. Further evidence for compartmentation of adenine nucleotides in cardiac myofibrillar and sarcolemmal coupled ATPase-creatine kinase systems

In isolated and purified cardiac myofibrillar and sarcolemmal preparations, the route of movement of ADP produced in the Mg2+-ATPase reactions was studied by investigating the efficiency of competition between the endogenous creatine kinase and exogenous pyruvate kinase reactions. In the homogeneous control system composed of hexokinase and glucose as ATPase, soluble creatine kinase rapidly rephosphorylated ADP produced in the presence of 1 mM ATP, but the addition of pyruvate kinase in an increasing amount inhibited the reaction of creatine release from phosphocreatine and symmetrically increased the rate of pyruvate production from phosphoenol pyruvate. At a pyruvate-kinase/creatine-kinase activity ratio (PK/CK) of 50, all ADP was used by the pyruvate kinase. In myofibrillar and sarcolemmal preparations containing particulate creatine kinase, the creatine kinase reaction was much less efficiently suppressed by pyruvate kinase, and at PK/CK = 50 half-maximal release of creatine was still observed. The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme. The physiological significance of these findings is discussed.     

Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: implications for the design of better anti-HIV analogues

The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the D-/L-configurations of the nucleotides, the L-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the L-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an L-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.     

Octameric mitochondrial and dimeric cytoplasmic creatine kinase. The number of subunits, participating in catalysis

It was found that in the octameric form of mitochondrial creatine kinase (Mr = 340 kD), only 52% of active centers bind Mg-ADP into a E-Mg-ADP-creatine complex with the dissociation constant, K(Cr)ADP, of 0.105 mM, which is close to the Km value for the enzyme (0.072 mM). In the dimeric form of cytoplasmic creatine kinase (Mr = 82 kD), 100% of active centers bind Mg--ADP; the K(Cr)ADP value (0.11 mM) is close to the Km value for the given enzyme preparation (0.083 mM). All active centers of rabbit muscle cytoplasmic creatine kinase were shown to form an analog of the transition state complex (ATSC) - E-Mg-ADP-NO3- -creatine. The constant for Mg-ADP dissociation from ATSC is identical for all centers of cytoplasmic creatine kinase and equals to 6.0 microM. The curves for ATSC saturation with Mg-ADP in the presence of iodacetamide for mitochondrial creatine kinase were constructed and computer analyzed. It was shown that in the octameric form of the enzyme only 54 +/- 13% of subunits can form ATSC. The constant for Mg-ADP dissociation from ATSC, KATSCADP is equal to 1.9 +/- 0.8 microM. It was concluded that 50% of subunits of the octameric form of mitochondrial creatine kinase are not involved in the catalytic act due to masking of their active centres and their inability to form transition state complexes. A model of regulation of cell supply with high energy compounds, e.g., ATP, creatine phosphate, via association-dissociation of mitochondrial creatine kinase oligomers is proposed.     

Specific enhancement of the cardiac myofibrillar ATPase by bound creatine kinase.

The kinetic influence of bound creatine kinase (CK) on the Ca(2+)-activated myosin ATPase was evaluated. ATPase rates were measured from 0.8 microM to 3.2 mM MgATP. Under control conditions, the apparent KmATP was 79.9 +/- 13.3 microM. In contrast, the addition of 12.2 mM phosphocreatine (PCr) decreased the apparent KmATP to a value of 13.6 +/- 1.4 microM. To determine if this reduction was merely the result of an ATP maintenance system, ATP was regenerated using either phosphoenolpyruvate and pyruvate kinase (PEP-PK), or PCr and soluble bovine cardiac CK. Data obtained with PEP + PK indicated an apparent KmATP of 65.5 +/- 7.3 microM. To study the effects of exogenous CK, the endogenous CK was irreversibly inhibited with 1 mM iodoacetamide. The kinetics of the ATPase were then examined by adding soluble CK to the incubation medium. Under these conditions, the KmATP was 56.4 +/- 0.86 microM. Therefore, these two ATP regeneration systems could not duplicate the effects of endogenous CK. The reduction of the apparent KmATP by endogenous CK was not the result of an altered inhibition by MgADP. MgADP inhibition was determined to be non-competitive, with a Ki of 5.0 +/- 0.1 mM. These data suggest that the observed kinetic effects reflect the proximity of the enzymes in the myofibrillar bundle, thus emphasizing the importance of bound CK for the localized regeneration of MgATP utilized by the myosin ATPase.     

PEI-cellulose thin-layer chromatography. Product studies of the creatine kinase and pyruvate kinase reactions

Detailed methods for polyethylenimine-cellulose thin-layer chromatographic determination of the reactants and products of the creatine kinase and pyruvate kinase reactions are presented. The technique has been developed as a highly sensitive method both for estimation of relative initial rates afforded by creatine and creatine analogs in the creatine kinase reaction and also for estimation of trace amounts of inorganic phosphate and ADP contamination in samples of ATP.     

Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.

We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.     

Relating structure to mechanism in creatine kinase

Found in all vertebrates, creatine kinase catalyzes the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine may be viewed as a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. Consequently, creatine kinase plays a significant role in energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction. First identified in 1928, the enzyme has undergone intensive investigation for over 75 years. There are four major isozymes, two cytosolic and two mitochondrial, which form dimers and octamers, respectively. Depending on the pH, the enzyme operates by a random or an ordered bimolecular mechanism, with the equilibrium lying towards phosphocreatine production. Evidence suggests that conversion of creatine to phosphocreatine occurs via the in-line transfer of a phosphoryl group from ATP. A recent X-ray structure of creatine kinase bound to a transition state analog complex confirmed many of the predictions based on kinetic, spectroscopic, and mutagenesis studies. This review summarizes and correlates the more significant mechanistic and structural studies on creatine kinase.     

Creatine kinase equilibrium and lactate content compared with muscle pH in tissue samples obtained after isometric exercise

Muscle biopsies taken from the musculus quadriceps femoris of man were analysed for pH, ATP, ADP, AMP, creatine phosphate, creatine, lactate and pyruvate. Biopsies were taken at rest, after circulatory occlusion and after isometric contraction. Muscle pH decreased from 7.09 at rest to 6.56 after isometric exercise to fatigue. Decrease in muscle pH was linearly related to accumulation of lactate plus pyruvate. An increase of 22mumol of lactate plus pyruvate per g of muscle resulted in a fall of 0.5pH unit. The apparent equilibrium constant of the creatine kinase reaction (apparent K(CK)) increased after isometric contraction and a linear relationship between log(apparent K(CK)) and muscle pH was obtained. The low content of creatine phosphate in muscle after contraction as analysed from needle-biopsy samples is believed to be a consequence of an altered equilibrium state of the creatine kinase reaction. This in turn is attributed mainly to a change in intracellular pH.     

Myofibrillar creatine kinase and cardiac contraction

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.     

Interaction of pyruvate kinase with isatin and deprenyl

The key glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [³H]isatin binding (K_D ～10 μM) which is inhibited by ATP (IC₅₀ 25 μM) and deprenyl (IC₅₀ 5 μM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and the immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.     

Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation

To define more clearly the interactions between mitochondrial creatine kinase and the adenine nucleotide translocase, the outer membrane of rat heart mitochondria was removed by digitonin, producing an inner membrane-matrix (mitoplast) preparation. This mitoplast fracton was well-coupled and contained a high specific activity of mitochondrial creatine kinase. Outer membrane permeabilization was documented by the loss of adenylate kinase, a soluble intermembrane enzyme, and by direct antibody inhibition of mitochondrial creatine kinase activity. With this preparation, we documented four important aspects of functional coupling. Kinetic studies showed that oxidative phosphorylation decreased the value of the ternary enzyme-substrate complex dissociation constant for MgATP from 140 to 16 microM. Two approaches were used to document the adenine nucleotide translocase specificity for ADP generated by mitochondrial creatine kinase. Exogenous pyruvate kinase (20 IU/ml) could not readily phosphorylate ADP produced by creatine kinase, since added pyruvate kinase did not markedly inhibit creatine + ATP-stimulated respiration. Additionally, when ADP was produced by mitochondrial creatine kinase, the inhibition of the translocase required 2 nmol of atractyloside/mg of mitoplast protein, while only 1 nmol/mg was necessary when exogenous ADP was added. Finally, the mass action ratio of the mitochondrial creatine kinase reaction exceeded the apparent equilibrium constant when ATP was supplied to the creatine kinase reaction by oxidative phosphorylation. Overall, these results are consistent with much data from intact rat heart mitochondria, and suggest that the outer membrane plays a minor role in the compartmentation of adenine nucleotides. Furthermore, since the removal of the outer membrane does not alter the unique coupling between oxidative phosphorylation and mitochondrial creatine kinase, we suggest that this cooperation is the result of protein-protein proximity at the inner membrane surface.     

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.     

Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action.

We recently identified glutamine:fructose-6-phosphate amidotransferase (GFAT) as an insulin-regulated enzyme in adipocytes. Moreover, we found that loss of GFAT activity is not due to a direct action of insulin but rather is mediated by enhanced glucose uptake and the subsequent routing of glucose through the hexosamine biosynthesis pathway. To assess whether other cytosolic enzymes are controlled through formation of hexosamine products, we treated adipocytes for 5 h with physiological concentrations of insulin (ED50 = 0.33 ng/ml), glucose (ED50 = 4.5 mM), and glutamine (ED50 = 4.4 mM) and then measured pyruvate kinase (PK) activity. Combined treatment resulted in a progressive (t 1/2 of 2.5 h) and marked (3-fold) increase in PK activity, whereas omission of one or more of these components failed to alter enzyme activity. Several lines of additional evidence implicated the hexosamine biosynthesis pathway in PK regulation; therefore, it appears that the M2 isoform of pyruvate kinase represents another enzyme regulated by insulin through stimulation of glucose uptake and formation of hexosamine products. Related studies revealed that enhancement of PK activity is dependent upon ongoing mRNA synthesis and de novo protein synthesis and is mediated by an increase in enzyme content. Considered together, these findings provide new insights into the cascade of metabolic events triggered by insulin and implicated a novel metabolic pathway in the pretranslational control of enzyme function.     

Inhibition of pyruvate kinase of bovine adrenal cortex by ATP: the role of magnesium]

The effect of ATP on bovine adrenal cortex pyruvate kinase has been studied. ATP is a competitive inhibitor of the enzyme, the Ki being 3.2 mM. Based on the efficiency of tryptophan fluorescence quenching, it was concluded that the magnesium complex of ATP is a true enzyme inhibitor. The role of Mg2+ in the inhibition process consists in the formation of a bridge between the enzyme and ATP. The ATP-dependent mechanism of pyruvate kinase inhibition is a potential physiological regulator of the enzyme determining the lower threshold of its sensitivity in vivo.     

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.     

Pyruvate kinase from Calicophoron ijimai trematodes and its potential inhibition by anthelmintic preparations

It was shown that pyruvate kinase (PK) in the supernatant fraction from Calicophoron ijimai is able to regulate the direction of metabolic flow at glucose break down from phosphoenolpyruvate (PEP) level. The enzyme for activity required substrate, dinucleotides, cations K+ and Mn++. The activity with Mg++ as divalent cation is low. The addition of fructose-1.6-diphosphate (FDP) did not affect the enzyme activity with Mn++, however, increased the affinity for PEP. The velocity of Mg++ activated reaction increased by 8.2 times in the presence of FDP. PK in C. ijimai is sensitive to ATP inhibition, being weakly inhibited by malate. L-alanine did not influence on the enzyme activity. The effect of some anthelminthic preparations on the PK activity was shown.     

Crystal structure of brain-type creatine kinase at 1.41 A resolution

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates.     

A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20,000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg-ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg-ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co-immobilized myosin and creatine kinase, without direct interaction or 'intimate coupling' between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.     

The phosphoenol-pyruvate branchpoint in adult Hymenolepis diminuta (Cestoda): a study of pyruvate kinase and phosphoenol-pyruvate carboxykinase.

The properties of pyruvate kinase (PK) and phosphoenol pyruvate carboxykinase (PEP CK), two enzymes that determine the preferrential accumulation of either succinate or lactate as endproducts of carbohydrate metabolism, are described in adult Hymenolepis diminuta. PK activity at Vmax and Km levels of PEP was unaffected by ATP, alanine, FDP4, OR H+ ions, but was inhibited by 50% at 6.3 mM L-lactate and 30 mM HCO3. The addition of 30 mM HCO3 increased the Km(PEP) by 6-fold but did not alter the Vmax. The inhibition of PK by HCO3 cannot be explained entirely by an effect of ionic strength, but probably represents a specific modulator-enzyme interaction. Under similar conditions PEP CK was maximally activated. Although L-lactate inhibited PEP CK (Ki(lac) = 1.8 mM), this effector may play a minor role in regulation of PEP flux. These results implicate the poise of the HCO3-:CO2 system as a major determiner of endproduct accumulation in H. diminuta.