Ternary Enzyme-substrate Complex Research Articles

Creatine kinase and protein kinase reactions of cardiac cell membranes

This report summarizes the results of our recent research on the creatine kinase isozymes associated with the cardiac mitochondrial and sarcolemmal membranes and also describes some unique properties of the sarcolemmal protein kinase reaction. In heart mitochondria, the kinetics of creatine kinase is influenced by oxidative phosphorylation, with alterations in the substrate dissociation parameters of the creatine kinase reaction being the dominant effect. The change is specific for the dissociation of ATP from the ternary enzyme-substrate complex, expressed by an order of magnitude decrease in K a . This tight binding of MgATP to the E·MgATP·Cr complex also limits the degree of product inhibition by phosphocreatine. Thus, the isozyme can catalyze the rapid synthesis of phosphocreatine even in the presence of physiological and inhibitory concentrations of this product. The results emphasize the importance of the functional coupling between creatine kinase and the adenine nucleotide translocase, which may be viewed as a potential multi-step complex integrated for the aerobic synthesis of phosphocreatine. With respect to cytoplasmic forms, creatine kinase MM isozyme bound to the cardiac sarcolemma effectively utilized phosphocreatine for energizing the active transport of sodium and potassium. Therefore, both mitochondrial and sarcolemmal creatine kinase reactions are clearly affected by their localization at membranes and by reactions in their intimate microenvironments. The sarcolemmal membranes also exhibited endogenous protein kinase activity, phosphorylating the membranes of the vesicles. Both the endogenous sarcolemmal protein, with a molecular weight of 11 500 daltons, and an exogenous protein (histone) were used as substrates for the phosphorylation. In contrast to the histone phosphorylation reaction, the endogenous phosphorylation reaction was insensitive to cAMP. Kinetic analysis of the protein kinase reaction associated with the sarcolemma, using histone and Mg γ- 32P-ATP as substrates, showed that this reaction may occur by a sequential, quasi-equilibrium Bi-Bi mechanism, characterized by the following kinetic constants: K m (MgATP) = 12.1 μ m; K m (histone) = 0.47 mg/ml; K i (MgADP) = 15.6 μ m. A comparison of the results of these experiments with data cited in the literature for soluble protein kinase indicated that these enzymes are similar with respect to their kinetic mechanisms.

Pressure relaxation studies of isomerisations of horse liver alcohol dehydrogenase linked to NAD + binding

1. Introduction In previous experiments we showed that proton liberation during the oxidation of ethanol by horse liver alcohol dehydrogenase (LADH) occurred during formation of the binary and ternary enzyme-substrate complexes rather than during the hydride transfer step [l] Furthermore this ionisation change is in some way linked to quenching of protein fluorescence, suggesting a change in the environment of one of the two tryptophans of the enzyme subunits [2,3]. From these experiments and further studies of the pH dependence of protein fluorescence a model was proposed for the conformational states of this enzyme [3,4]. In order to account for the different pK, values for ADP-ribose equilibrium binding (8.9) and the NAD’ binding rate (9.8) Shore et al. [4] proposed that two conformations of the enzyme exist at neutral pH, EH and gH. The latter form showed quenched protein fluorescence, and was linked to the ionization of an enzyme functional group with a pK, less than 9. The scheme proposed to account for this is similar to our current mechanism. By the use of relaxation techniques in the present study, it has been possible to demonstrate directly the existence of these confor- mational states and the pH linkage. Coenzyme binding markedly slows down the relaxation of the confor- mational equilibria. 2. Experimental LADH was prepared by the method of Theorell et al. [5] and the concentrations in terms of one

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.

2-p-toluidinylnaphthalene-6-sulfonate, a fluorescent reporter group for L-isoleucyl-tRNA synthetase

The binding of 2-p-toluidinylnaphthalene-6-sulfonate to L-isoleucyl-tRNA synthetase is associated with a strong increase of fluorescence intensity and a concomitant shift in the wavelength of maximum emission. Binding of the fluorophore does not interfere with substrate binding nor the ATP- 32PP i exchange activity of the enzyme. The formation of binary and ternary enzymesubstrate complexes is indicated by a significant quenching of the fluorescence. Dissociation constants were determined from the degree of quenching as a function of the substrate concentration. Evaluation of spectrofluorometric data suggest that substrate binding is associated with a conformation change in which tryptophan side chains of the protein are involved.

Kinetic of the protection of lactate dehydrogenase by substrates against heat inactivation

1. 1. NAD +, NADH, lactate, pyruvate and the substrate analogues oxamate and oxalate all protected pig lactate dehydrogenase (EC 1.1.1.27) isoenzymes M 4 and H 4 against inactivation at 52–64° in Tris-Chl buffer (pH 7.5). The time-course of inactivation could be described as a single first-order reaction, both in the presence and absence of substrates. Protection by substrates was dependent on substrated concentration. 2. 2. For kinetic treatment of the data, protection was defined as the difference between the first-order rate constants of inactivation measured in the absence and in the presence of substrate ( Δk), respectively. On the assumption that protection was due to the equilibrium formation of enzyme-substrate complexes, simple equations were derived for calculation of the dissociation constants involved. A convenient procedure was found for distinguishing between the random order and compulsory order formation of a ternary complex. 3. 3. Protection, defined as Δk, was a hyperbolic function of the concentration of NAD + and NADH indicating the expected proportionality between protection and coenzyme binding. The dissociation constants calculated from these hyperbolic functions were of the order of 1·10 −5-1·10 −4 M which, in turn, conformed with the idea that protection was due to binding of coenzyme to the specific binding sites. Lactate, pyruvate, oxamate and oxalate, when added to lactate dehydrogenase in the absence of coenzyme and at concentrations lower than about 1·10 −2 M, did not protect lactate dehydrogenase against heat inactivation. The protection observed at higher concentrations was most marked with oxalate and it was definitely a nonhyperbolic function of oxalate concentration. 4. 4. Lactate, pyruvate, oxamate and oxalate, when added to lactate dehydrogenase in the presence of coenzyme, had a marked protective effect at concentrations as low as 1·10 −4 M. Detailed investigations were carried out with the lactate dehydrogenase-H 4 + NAD + + pyruvate ternary system. Assuming that protection was due to the equilibrium formation of a ternary enzyme-substrate complex, an attempt was made to determine the reaction sequence and to calculate the dissociation constants involved. By applying the above kinetic equations, we foudn that the ternary complex was formed in a compulsory order, with pyruvate being bound only to the lactate dehydrogenase-NAD + complex and not to the free enzyme. A dissociation constant of 0.75·10 −3–0.96·10 −3M was calculated for pyruvate in the ternary complex, whilst the dissociation constant obtained for NAD + was 1.5·10 −3M.

Neuere Ergebnisse unserer Forschung über Stärke

More Recent Results of our Research on Starch.The authors most important research results of the last decade have been compiled. Among those there are first of all works on the course of the starch degradation process as well as on the method for measuring molecular distribution, which had been specially designed for this purpose. As to the processes involved in hydrolytic starch degradation by acid, respectively amylases, mainly the different cleavage probabilities of the glucoside bonds have been examined. Determination of the kinetic constants during amylolytic degradation of starch added to elucidate the molecular degradation mechanism. Especially the ß‐amylolysis of a special amylose marked by 14C has been examined more closely.The second research field consisted of the determination of the functional groups of potato phosphorylase, of the functional groups of the substrate as well as possible binary and ternary enzyme substrate complexes. On the basis of this theoretical enzyme research an enzymatic brewing technology has been developed, which is already being applied in several production plants.

Kinetic Studies of Hypoxanthine-Guanine Phosphoribosyltransferase

Abstract The mechanism of reaction of human erythrocyte hypoxanthine-guanine phosphoribosyltransferase was investigated by initial velocity, product inhibition, and isotope exchange studies. Although initial velocity data are compatible with a mechanism involving binary enzyme-substrate complexes, the product inhibition and isotope exchange studies indicate that the reaction is ordered with the formation of ternary enzyme-substrate complexes. Product and alternative product inhibition data, alternative substrate inhibition experiments, and combined alternative substrate initial velocity data are all consistent with the view that only one enzyme catalyzes this reaction of hypoxanthine and guanine, and that they share a common binding site.