Specific ligand-induced association of an enzyme. A new model of dissociating allosteric enzyme

The kinetic behavior of dissociative enzyme system of the type inactive monomer in equilibrium active dimer where dimeric form is stabilized by specific ligand (in particular by substrate) which is bound in the region of the contact of monomers has been analysed. It is assumed that the dissociation of dimer results in formation of monomers which retain the subsites for specific ligand binding. The shape of the dependences of enzyme reaction rate (v) on substrate concentration (S) has been characterized using the order of enzyme reaction rate with respect to substrate concentration: ns = d ln v/d ln [S]. When the substrate concentrations are low the dependences of v on [S] have S-shaped form (the maximum value of ns exceeds the unity) at the definite values of the parameters of the enzyme system. The value of ns approaches--2 at sufficiently high substrate concentrations (in the region where the substrate reveals the inhibitory effect due to blocking the association of inactive monomers into active dimer). The methods of calculation of the parameters of the dissociative enzyme system under discussion have been elaborated on the basis of the analysis of the experimental dependences of specific enzyme activity on enzyme concentration obtained at various fixed substrate concentrations.     

Study of kinetics of thermal aggregation of mitochondrial aspartate aminotransferase by dynamic light scattering: protective effect of α-crystallin

Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster–cluster aggregation. Suppression of mAAT aggregation by α-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster–cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.     

Supramolecular organization of glycolytic enzymes

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.     

Stationary kinetics of the function of multienzyme membrane sensors with electrochemical enzyme regeneration]

A theoretical analysis of the functioning of membrane biosensors based on consecutive polyenzymatic transformations of the substrate and detectable by indicator electrodes with electrochemical regeneration of the enzyme active sites, has been carried out. Correlations between the substrate concentration, diffusion-catalytic characteristics of the coating components, and rate of the indicator reaction on the one hand, and the level of the steady-state response of the polyenzymatic biosensors, on the other, have been determined for relatively low values of the polyenzymatic layer thickness. Various regimens of functioning of bienzymatic electrodes have been considered.     

The method of Hill's coefficient calculation for the region of inhibition of an enzyme by excess of the substrate. Inhibition of phosphofructokinase by excess of ATP]

The new method for Hill's coefficient (nH) calculation in the region of substrate concentrations where the latter acts as an inhibitor has been developed. The method does not need preliminary determination of maximum value of enzyme reaction rate (V) for ascending branch of the plot of enzyme reaction rate versus substrate concentration and allows to avoid over-estimation of value of nH when the magnitude of optimal reaction rate is less than value of V. The literature data for inhibition of phosphofructokinase by excess of ATP are used for illustration of applicability of the suggested method of Hill's coefficient calculation.