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E A Saburova

Publications and source records attributed to E A Saburova.

16 recordsLinked to original sources
PubMed2000

Inhibitory effect of polyelectrolytes on oligomeric enzymes.

The effect of polyelectrolytes on the stability and catalytic characteristics of oligomeric enzymes--pig muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GDH)--was studied by fluorescent spectroscopic and steady state kinetic methods. It was shown that the binding of negatively charged polyelectrolytes--polystyrene sulfonate, polymethacrylate, and polyphosphate--destroys the tertiary and partially the secondary structure of LDH and GDH, resulting in their complete inactivation at pH < 7. The concentrations of polyelectrolytes needed for inhibition of the enzymes were in this case by two or more orders of magnitude lower than the corresponding concentrations for monomers--toluene sulfonate, methacrylate, and phosphate. The affinity of the substrate (pyruvate) for LDH did not vary in the presence of the polyelectrolytes, but the inhibition was removed by excess of substrate. We propose that the oligomeric state of enzymes causes polyelectrolytes to act on them in a special manner, this special effect differing significantly from the effect of polyelectrolytes on monomeric enzymes. The effect consists in that polyelectrolytes cleave the oligomeric structure of the enzymes, this "cleaving" effect being higher the greater the hydrophobicity of the polyelectrolyte chain.

Animals↗
PubMed1990

[Kinetic study of the mechanisms of eliminating substrate inhibition of lactate dehydrogenase by anions and pH].

The dependence of lactate dehydrogenase inhibition at high pyruvate concentrations on pH and neutral salt anions was studied. It was shown that Cl- anions compete with the substrate within the ternary inhibitory complex, ENADpyr in equilibrium ENADCl-, as a result of which the pyruvate-induced inhibition is eliminated. The KD values for Cl- (50 mM) and I- (27 mM) were calculated from the substrate velocity curves at high concentrations of pyruvate. It was supposed that pyruvate inhibition elimination by OH- proceeds via the same kinetic mechanism. The pK value (7.1 +/- 0.1) calculated from this model corresponds to pKn of essential His-195. The additivity of OH- and Cl- function was demonstrated.

Animals↗
PubMed1989

Kinetics of the lactate dehydrogenase reaction in high-viscosity media.

The effect of the medium viscosity on kinetics parameters of lactate dehydrogenase reaction was studied. The viscosity increase results in a sharp decline in the catalytic rate for both the pyruvate reduction and lactate oxidation reactions. It is shown that the catalytic step and its associated conformational motions is the only step which is considerably retarded when the viscosity increases. The reaction is not sensitive to changes in the dielectric properties of the medium. An inverse power function observed between the rate constant and viscosity cannot be explained by the theory of absolute reaction rates. However, it can easily be interpreted on the basis of the Kramers theory dealing with the transition over the activation barrier as a diffusional motion in the field of random forces. The influence of the medium's viscosity on the kinetic parameters indicates the existence of strong coupling between the dynamics of the solvent and the conformational motions of the protein molecule, which are correlated with the catalytic step.

Animals↗
PubMedDate not supplied

[Lactate dehydrogenase in an interpolyelectrolyte complex. Function and stability].

A new method for encapsulating enzymes by multilayer polyelectrolyte coating is proposed. The method consists in a stepwise adsorption of polyelectrolytes from solution onto protein aggregates formed by salting out the proteins in highly concentrated salt solutions. Polystyrene sulfonate and fluorescence-labeled polyalylamine were used for capsule formation. The size of lactate dehydrogenase aggregates covered by four layer pairs of electrolytes was 1-5 microns, as indicated by fluorescence microscopy. The catalytic characteristics and stability of pig muscle lactate dehydrogenase (EC 1.1.1.13) incapsulated in multilayer electrolyte complex obtained by this method were studied. It was found that the affinity of the substrate pyruvate for the enzyme in the polyelectrolyte complex (K(M)) did not essentially change as compared with the free enzyme. Incapsulated lactate dehydrogenase showed the following features that distinguish it from the free form: (1) the lifetime in diluted solutions increases from 30 min (without capsules) to 1-2 days (in capsules); (2) a higher stability to basic denaturation (up to pH 10); and (3) the absence of substrate inhibition of enzyme in the polyelectrolyte complex. The changes in the catalytic characteristics of incapsulated lactate dehydrogenase are discussed in terms of an increase in effective pK values of amino acid perturbed by polyelectrolyte coating of enzyme.

Animals↗
PubMedDate not supplied

[Effect of anions on inhibition of lactate dehydrogenase by pyruvate].

The effect of anions Cl- and I- on structural and kinetic properties of LDH was investigated. It was shown that anions are specific inhibitors of LDH competing with pyruvate in the active ternary complex, LDHNADHpyq. The following dissociation constants for the anions were obtained from inhibition data: 0.4 +/- 0.02 and 0.07 +/- 0.01 M for Cl- and I-, respectively. The slope of Hill plot are near 1.0. The anions abolished the inhibition of LDH at high pyruvate concentrations. The following dissociation constants were obtained from these data: 0.1 and 0.015 M for Cl- and I- respectively. The inhibition by anions and the abolishing of substrate inhibition by anions were studied also for the lactate oxidation reaction. The dissociation constants for anions obtained from these data are in good correlation with the constants obtained for the pyruvate reduction reaction. It was concluded that anions do not interact with the group at the catalytic site with pK approximately 7.8, presumably His-195. The degree of pyruvate inhibition does not depend on the buffer system. The differences in the degree of inhibition obtained previously in phosphate, imidazole and tris-buffer systems can be explained by the presence of Cl- anions in the last two buffer. The rate constants of hydroxy leads to keto pyruvate transition was obtained in various buffer systems. It was shown that the hydroxy-form of pyruvate does not cause the inhibition of LDH.

Animals↗
PubMedDate not supplied

[Dynamic aspect of kinetics of reaction catalyzed by lactate dehydrogenase].

The influence of solvent viscosity on the kinetic parameters of the pyruvate reduction reaction catalyzed by lactate dehydrogenase has been investigated. The viscosity was adjusted by sucrose and glycerol solutions at concentrations from 0 to 44% and from 0 to 63%, respectively. The reaction rate decreased abruptly with an increase in viscosity. The study of different reaction stages (enzyme-substrate complex formation, catalysis, inhibitory complex decomposition, competitive inhibition by chlorine ions) revealed that the catalysis (and the related conformational changes) is the only stage (of the above mentioned) that depends markedly on the solvent viscosity. The reaction is insensitive to the changes in the dielectric properties of the solution induced by the addition of alcohols and dioxane. The observed power dependence of the rate constant on viscosity is explained in terms of Kramer's theory which considers the proton transition through the activation barrier to be a diffusion in the field of random forces. The influence of solvent viscosity on enzymic kinetics indicates a direct relation between solvent dynamics and relevant protein conformational movements.

Animals↗
PubMedDate not supplied

[pH-dependence of the structural and functional properties of lactate dehydrogenase (M4). Indirect cooperativity in lactate dehydrogenase].

The dependence of structural and functional properties of LDH on pH in the 6.0--9.0 region was investigated. There were no marked deviations of pyruvate reduction initial velocity curves from the Michaelis--Menten equation in a wide range of pyruvate concentrations. It was shown that Vmax changes negligibly in the 6.0--9.0 pH regions, but Km increased markedly with pH elevation. The pK value of 7.8+/-0.1 was obtained for 50% changes of pyruvate binding. The dependence of enzyme inhibition from pH at a high pyruvate concentration (20 mM) was investigated. At pH values above 8.0 pyruvate inhibition disappeared. The dependence of the inhibition degree from pH was estimated as pK 7.8+/-0.1. Hill coefficient (n) calculated from the curves of Km and the degree of substrate inhibition depending on pH was 1.6; n for pyruvate inhibition at pH 7.5 was 2 greater than n greater than 1 for moderate substrate concentrations (1--5 mM) and n approximately 1 for higher concentrations (5--40 mM). The value of n approximately 1 at pH 7.8 was obtained. The model suiting all available data concerning the cooperativity phenomena in LDH during protonation and inhibition by pyruvate is outlined. The model is based on the results indicating the slow isomerisation of LDH in ternary complexes with NADH and pyruvate and the absence of equilibrium on the intermediate stage of reaction.

Animals↗
PubMedDate not supplied

[Biphase reaction of enzymatic pyruvate reduction during inhibition].

At least two stages have been revealed in the reaction of enzymatic reduction of pyruvate under inhibition. A fast process during the first stage of the reaction was finished in a period of "dead time" with routine spectroscopic measurements. A reaction rate at this stage decreased by three times. The second stage was characterized by a constant rate, which changed by less than 10%. The analysis of neutral salt influences on the inhibition complex showed, that the latter decomposed rapidly. A model suggested earlier, was used for interpreting the reversible product inhibition of the reaction catalysed by LDH.

Animals↗
PubMedDate not supplied

[Non-linear, non-competitive inhibition of lactate dehydrogenase with carboxylic acid anions (substrate analogues)].

The carboxylic acid anions are shown to inhibit lactate dehydrogenase in a non-competitive manner at pH 8.5. The inhibitory effect of different carboxylates used increases in the direction acetate leads to formate leads to methacrylate. Non-competitive inhibition by carboxylates has a non-linear character with effective Hill exponent depending on the inhibitor concentration increasing to the value n approximately equal to 2 when inhibitor concentration increases. The kinetic model is proposed to account for non-linear non-competitive inhibition in consequence of the abortive ternary complexes formation in two enzyme conformations. The substrate analogues properties that result in non-competitive inhibition of lactatedehydrogenase are considered.

Animals↗
PubMedDate not supplied

[Regulation of two-substrate reaction by substrate analog, as analyzed by graphic methods].

The regulatory action of a substrate analogue on the two-substrate reaction has been analysed by the method of graphs. It was shown that the analogue can act not only as inhibitor but also as activator in an ordered mechanism of the two-substrate reaction. Activation of the reaction by the analogue is a consequence of its random mechanism in regard to substrates and products. This random mechanism tends to the appearance of another route in the enzymic reaction and by this route the activation of the reaction may be obtained. It was also shown, that substrate inhibition may be abolished by the analogue, if the reaction rate via analogue route is large enough. On this case the analogue competes with the substrate-inhibitor in the non-active ternary complex. For the analysis of kinetic schemes the cyclic algorithm of the graph method was applied because of its convenience for multiroute reactions. Some simplifications were proposed for the structure of graphs. The results of this analysis are applicable for interpretation of abolishing the substrate inhibition by monovalent anions for some dehydrogenases.

Enzymes↗