Thermokinetic Studies On Activation And Inhibition Of Enzyme-catalyzed Reaction

Basis on the basic principle theory and method of thermokinetics, four enzymatic reaction systems, i.e. catalase, arginase, horseradish peroxidase and acetylcholinesterase and there inhibitory and activation kinetics have been studied by LKB-2107 BATCH microcalorimeter in this thesis. The studies on each enzyme are listed below:I. Thermokinetic study on the catalase-catalyzed reaction1. Basis on Chance mechanism of catalase-catalyzed reaction, the observed first reaction rate constant and second rate constant has been determined. At 37 and pH 7.0 phosphate buffer solution, the observed second rate constants was determined to be 7.32x106 L-mo1-1s-1.2. The reversible inhibition of catalase-catalyzed reaction by NaN3 was studied. In the reaction system, NaN3 was joined into the slow react cycle and made the compound I, which has enzymatic activity, changed to compound II, one style of enzyme with no enzyme activity, and then decreased the reaction activation of catalase. The half inhibitory concentration was determined to be 8.5lxl0-6nol-L-1 in this experiment.3. L-ascorbic acid and Cu2+ cooperating inhibition on catalase-catalyzed reaction was studied using thermokinetic reduced extent method. There were no apparent inhibit effect on catalase when L-ascorbic acid and Cu2+ were presence solely, but when they were presence simultaneously, they can non-linear inhibit the reaction. The dose relation of enzyme activity with the concentration of L-ascorbic and Cu2+ were established as a sigmoidal curve. Combine the results for studying on the natural oxidation of L-ascorbic acid in literature, the oxidation intermediate of L-ascorbic acid, AsA, was suggested to be the real inhibitor of catalase, and the dose relation of enzymatic activation with the concentration of L-ascorbic and Cu2+ implicate the relation of concentration of the intermediate and the concentration of L-ascorbic and Cu2+.II. Thermokinetic study on the activation of arginase-catalyzed reactionThe activation effects of exogenous manganese ions and L-glycin on arginase-catalyzed reaction were studied using thermokinetic reduced extent method. The experiments explicated that full activation of arginase, by incubation with appropriate concentration of Mn2+, resulted in increased Vmm and a higher sensitivity of the enzyme to product and L-lysine inhibition, with no change in the Km for arginine. But appropriate concentration of glycin can enhance the activity of arginase, one possible activation mechanism of arginase by glycin was suggested that the activation effect is result from the competition of glycin and arginine to enzyme activity position, when one or two of the activity position of arginase is occupied by glycin, it is propitious of the enzyme to complex with substrate and obstruct L-ornithine combine with enzyme, when all of the activity position are occupied by glycin, the activation effect vanish and the inhibition effect appear. With the increase of the concentration of manganese ions and L-glycin, This experimenthas also discovered that there is a maximum values in the increasing of the enzymatic reaction velocity with the increase of the concentration manganese ions and L-glycin. When the activator excesses, the velocity of enzyme reaction decreased. For manganese ions, it must be the reason that the manganese ions were oxided to other ions forms such as Mn(fll), and these forms would inhibit the arginase reaction; but for L-glycin, it must be resulted from more than one of glycin molecular combined with arginase, and it obstructed the combine of the substrate and enzyme, and then decreased the activity of the enzymatic reaction. HI. Thermokinetic studies on the inhibition of arginase-catalyzed reaction1. The inhibition of the reaction by I-lysine was studied at 37, arid pH 9.4 sodium barbiturate-HCl buffer solution. The inhibitory constant (K\) of Z,-lysine was determined to be 5.6 xl0"3mol-L"' and 3.10 xiO"3mol-L~! in the absence of exogenous manganese ions and in the presence of saturated concentration of Mn2+, respecti...