KINETIC STUDIES OF THE REVERSE REACTION CATALYSED BY ADENOSINE TRIPHOSPHATE-CREATINE PHOSPHOTRANSFERASE. THE INHIBITION BY MAGNESIUM IONS AND ADENOSINE DIPHOSPHATE.
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ABSTRACT:
SUBMITTER: MORRISON JF
PROVIDER: S-EPMC1206431 | biostudies-other | 1965 Jan
REPOSITORIES: biostudies-other
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Project description:1. The forward and reverse reactions catalysed by ATP-creatine phosphotransferase have been studied kinetically at pH8.0 in the presence and absence of products, under conditions in which the free Mg(2+) concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP(2-)+creatine(0)right harpoon over left harpoonMgADP(-)+phosphocreatine(2-)2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme-MgADP-creatine and enzyme-MgATP-phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme-MgADP-creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme-MgATP-phosphocreatine complex, where each reactant hinders the combination of the other.
| S-EPMC1264541 | biostudies-other
Project description:1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.
| S-EPMC1179640 | biostudies-other
Project description:The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K'i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 X 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.
| S-EPMC1164896 | biostudies-other
Project description:1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP(2-) based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP(2-) complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP(2-) complex and to the presence of the inhibitory free uncomplexed ATP(4-) concentration are also explained in terms of a non-allosteric role for either Mg(2+) or MgATP(2-) fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis-Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal-substrate complex.
| S-EPMC1174301 | biostudies-other
Project description:The progression of pressure-overload left ventricular hypertrophy (LVH) to chronic heart failure (CHF) may involve a relative deficit in energy supply and/or delivery.We measured myocardial creatine kinase (CK) metabolite concentrations and adenosine triphosphate (ATP) synthesis through CK, the primary energy reserve of the heart, to test the hypothesis that ATP flux through CK is impaired in patients with LVH and CHF. Myocardial ATP levels were normal, but creatine phosphate levels were 35% lower in LVH patients (n = 10) than in normal subjects (n = 14, P < 0.006). Left ventricular mass and CK metabolite levels in LVH were not different from those in patients with LVH and heart failure (LVH+CHF, n = 10); however, the myocardial CK pseudo first-order rate constant was normal in LVH (0.36 +/- 0.04 s(-1) in LVH versus 0.32 +/- 0.06 s(-1) in normal subjects) but halved in LVH+CHF (0.17 +/- 0.06 s(-1), P < 0.001). The net ATP flux through CK was significantly reduced by 30% in LVH (2.2 +/- 0.7 micromol x g(-1) x s(-1), P = 0.011) and by a dramatic 65% in LVH+CHF (1.1 +/- 0.4 micromol x g(-1) x s(-1), P < 0.001) compared with normal subjects (3.1 +/- 0.8 micromol x g(-1) x s(-1)).These first observations in human LVH demonstrate that it is not the relative or absolute CK metabolite pool sizes but rather the kinetics of ATP turnover through CK that distinguish failing from nonfailing hypertrophic hearts. Moreover, the deficit in ATP kinetics is similar in systolic and nonsystolic heart failure and is not related to the severity of hypertrophy but to the presence of CHF. Because CK temporally buffers ATP, these observations support the hypothesis that a deficit in myofibrillar energy delivery contributes to CHF pathophysiology in human LVH.
| S-EPMC1808438 | biostudies-literature
Project description:1. Starch-gel analysis of extracts from adult human muscle, heart and brain reveals a hybrid creatine kinase with an abnormally high electrophoretic mobility. 2. Hybridization in vitro confirms that the postulated hybrid is formed from a combination of brain- and muscle-type enzyme sub-units. 3. The relative electrophoretic mobility of the hybrid is not affected by changing the starch concentration in the gel or by the buffer system used, or by electrophoresis in thin layers of Sephadex. 4. It is concluded that hybrid formation results in a net increase on the dimeric enzyme of from 4 to 6 negative charges. 5. During development a sharp increase in the rate of production of muscle-type enzyme sub-units occurs in heart at 10-13 weeks' gestation and in muscle at 18-20 weeks' gestation. The latter change is accompanied by a relative decrease in the concentration of brain-type sub-units.
| S-EPMC1198849 | biostudies-other
Project description:1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl(-) restores protection to the original value. The quaternary MgADP-creatine-enzyme-nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V(max.) and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.
| S-EPMC1174012 | biostudies-other
Project description:1. Substrate-saturation curves of brain hexokinase for MgATP(2-) were sigmoidal at sub-saturating concentrations of glucose when the Mg(2+)/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg(2+) was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg(2+)/ATP ratio 1:1, and 1.0 with excess of Mg(2+). The apparent K(m) for MgATP(2-) is 6.5x10(-4)m at a Mg(2+)/ATP ratio 1:1, and 3.5x10(-4)m with excess of Mg(2+). 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis-Menten kinetics at a Mg(2+)/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg(2+)/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg(2+). 4. High concentrations of Mg(2+) (Mg(2+)/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP(2-) and that Mg(2+) is an allosteric activator. Possible implications in regulation are discussed.
| S-EPMC1178047 | biostudies-other
Project description:The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.
| S-EPMC1168088 | biostudies-other
Project description:1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.
| S-EPMC1187779 | biostudies-other