Search

A study of reaction mechanisms of indole-3-acetate degradation by various peroxidases, in the absence of added hydrogen peroxide, has been performed employing stopped-fow and lowtemperature spectroscopic techniques. At pH 6 and for high [substrate]/[enzyme] ratios, peroxidase compound II is formed and is stable for several minutes. However, this enzyme compound becomes visible only after the transient appearance of ferroperoxidase and of compound III. This reaction process is apparently linked with the formation of 3-methyleneoxindole as the major end-product of indole-3-acetate degradation. On the other hand, when indole-3-acetate and peroxidase in stoichiometric concentrations are mixed at pH 4, only ferroperoxidase and compound III are formed and the process appears to be linked with the production of indole-3-aldehyde. Stopped-flow experiments show that indole-3-acetate is rapidly bound on peroxidase, this rapid binding step being followed by a slow one-electron transfer between indole-3-acetate and peroxidase. This reduction of ferriperoxidase is blocked at low temperature (-20 °C), whereas in these experimental conditions, peroxidation of indole-3-acetate in the presence of added hydrogen peroxide still occurs. Moreover, when compound III reacts with indole-3-acetate at low temperature (-15 °C}, this compound is regenerated as long as oxygen is present in the reaction medium. Neither ferroperoxidase nor compound I appear during this process. Most, if not all of the compound III is formed by oxygenation of the ferrous peroxidase. This compound III can react with an equimolar amount of indole-3-acetate to give ferrous peroxidase and indole-3-aldehyde. Moreover, compound III can react with two indole-3-acetate molecules leading to compound II formatiom. Because only small amounts of the organic peroxide derived from indole-3-acetate are present in the reaction medium, most of the compound II is formed by a two-equivalent reduction of... [ABSTRACT FROM AUTHOR]

Presented here are the optical characteristics (absorbance, fluorescence, circular dichroism) of several enzyme · coenzyme complexes of L-glutamate dehydrogenase, with and without the different substrates (or products). Three of the four complexes studied can be distinguished unambiguously by the following characteristics: the E · NADPH and E · NADPH · L-glutamate complexes have similar absorption and fluorescence properties, but markedly differ in their opposite ellipticities; the E · NADPH · 2-oxoglutarate possesses characteristic absorption and elliptical properties, but its highly quenched fluorescence is its most distinctive feature. A detailed study of the elementary kinetic parameters, k+ and k-, was carried out with respect to each ligand. A comparison is made of the Kd values calculated from the k-/k+ ratio and from direct binding studies. As far as NADPH is concerned it is clear that its association rate constant is not affected, while its dissociation rate constant is very sensitive to the presence of the substrates. Thus, the stability of each complex is mainly governed by the dissociation rate. These dissociation rate constants are not very different from those obtained for the different substrates. [ABSTRACT FROM AUTHOR]

Kinetic studies were carried out on the reaction mechanism of rat liver acetyl coenzyme A carboxylase. The results of initial velocity and product inhibition studies on the forward and reverse reactions indicate that the carboxylase reaction is most likely to proceed through the "bi bi uni uni ping pong" mechanism. The order of the addition of substrates to the enzyme is as follows: ATP, HCO3- and acetyl-CoA in the forward reaction, and malonyl-CoA, Pi and ADP in the reverse reaction. Kinetic analysis of the stimulatory effect of citrate on the initial velocities of the forward and reverse reactions indicate that the site of citrate action during catalysis lies predominantly on the carboxylated enzyme. It is proposed that there exists an equilibrium between active and inactive forms of the carboxybiotinyl enzyme and that this equilibrium is shifted toward the active form in the presence of citrate. [ABSTRACT FROM AUTHOR]

1. The initial rate of glyceraldehyde-P formation catalyzed by the tryptophan synthetase of Escherichia coli from indole-3-glycerol-P has been measured as a function of the concentrations of indole-glycerol-P, serine, tryptophan, and indole. The conversion of indole-glycerol-P to indole and glyceraldehyde-P is proposed to proceed by the ordered sequential release from the enzyme of indole followed by glyceraldehyde-P. Tryptophan does not appreciably inhibit this reaction. 2. Tryptophan formation is proposed to occur by the random sequential addition of serine and indole-glycerol-P to the enzyme. Tryptophan is a product inhibitor of the reaction, apparently combining with the free enzyme and also forming an enzyme-(indole-glycerol-P)-tryptophan dead-end complex. 3. Indole inhibits non-competitively with respect to both indole-glycerol-P and serine, and is proposed to combine with the free enzyme and with the enzyme having bound indole-glycerol-P, serine, or both substrates. 4. The implications of the results for the catalytic center and subunit structure of the enzyme are discussed. [ABSTRACT FROM AUTHOR]