Kinetic characteristics of L-lysine α- oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D: Substrate specificity and allosteric effects.

The present work aims to investigate the kinetic characteristics of homodimer enzyme L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D, taking into account allosteric effects. The enzyme was first shown to reveal positive cooperativeness, h=2.05±0.15. Using additional opportunities of Hill coefficient the value of the Michaelis-Menten constant has been estimated, Km=1.015∙10-5М, indicating high strength of substrate binding to the active site of each subunit. High selectivity and absolute L-stereospecificity of the enzyme were shown. The inhibition of L-lysine conversion by non-cleavable lysine analogs as well as the reaction product was found out to take place. These effects have been evaluated only as the inhibition coefficients (%). A more detailed study of these inhibition effects was complicated because of the cooperativeness of enzyme subunits mentioned above. The kinetic scheme of L-lysine α-oxidase was proposed involving parallel-subsequent action of each of two subunits in the catalytic act. We think that the results obtained will be useful for studying the kinetic properties of other multi-subunit enzymes and improve understanding of the mechanisms of their action.

Gossypol Inhibits Electron Transport and Stimulates ROS Generation in Yarrowia lipolytica Mitochondria.

This work studied the effect of gossypol on the mitochondrial respiratory chain of Yarrowia lipolytica. The compound was shown to inhibit mitochondrial electron transfer and stimulate generation of reactive oxygen species. The inhibition kinetics in oxidation of various substrates (NADH, succinate, α-glycerophosphate and pyruvate + malate) by isolated mitochondria was investigated. Analysis of the kinetic parameters showed gossypol to inhibit two fragments of the mitochondrial electron transfer chain: a) between coenzyme Q and cytochrome b with K(IIIi) of 118.3 µM (inhibition by the noncompetitive type), and b) at the level of exogenous NADH dehydrogenase with of K(Ii) 17.2 µM (inhibition by the mixed type).

Perspectives of data analysis of enzyme inhibition and activation, part 4: equations for calculation of constants of enzyme activation and inhibition.

Equations for calculation of constants of enzyme activation and nontrivial types of enzyme inhibition, which are not reported in the literature, have been deduced. Examples of using these equations are also presented.

Perspectives of data analysis of enzyme inhibition and activation, Part 1: Use of the three-dimensional K'm V'I coordinate system for data analysis of enzyme inhibition and activation.

The possibility of construction of the three-dimensional (unfolded and folded) K'm V'I rectangular coordinate systems convenient for vector representation of inhibited and activated enzymatic reactions as well as of a two-dimensional K'm V' scalar rectangular coordinate system convenient for diagrammatic representation of enzymatic reactions is considered. The perspectives of using the properties of the three-dimensional L vectors and their scalar L projections for data analysis of enzyme inhibition and activation are analyzed.

Perspectives of data analysis of enzyme inhibition and activation, Part 2: Parametrical classification of types of enzymatic reactions.

A parametrical classification of types of enzymatic reactions that counts 15 types-7 inhibited reactions, 7 activated ones, and 1 type of initial (uninhibited and nonactivated) reaction-is considered. The system permits taking into account the number of parameters of enzymatic reactions and symmetricity of a course of their change.

Perspectives of data analysis of enzyme inhibition and activation, Part 3: Equations for calculation of the initial rates of enzymatic reactions.

Equations for calculation of the initial rates of activated enzymatic reactions and the inhibited enzymatic reactions, unavailable in experimental enzymology, were obtained. Examples of practically using of these equations are given.