Activity and spectroscopic properties of bovine liver catalase in sodium bis(2‐ethylhexyl)sulfosuccinate/isooctane reverse micelles

Abstract

Spectroscopic properties such as ultraviolet-visible spectroscopy, circular dichroism, steady-state fluorescence and the catalatic activity of bovine liver catalase (BLC) have been studied in reversemicelles microemulsions formed by sodium bis(2-ethy1hexyl)sulfosuccinate (AOT) in isooctane. Activity measurements were carried out with the highly water soluble hydrogen peroxide as substrate as a function of temperature and pH, at various concentrations of substrate, enzyme, AOTThe folding of catalase in reverse micelles is not drastically altered, although there are changes in the local surrounding of the prosthetic group. Kinetic measurements have been carried out under first-order conditions at low substrate concentration. They indicate that the measured substrate decomposition rate is limited by the exchange of substrate molecules between the substrate-filled and the enzyme-containing micelles. The specificity constant, k, = k,,,/K,, a$ related to water pool concentrations, k:",,, was, at all wo values, considerably lower than k, measured in buffer (k:). In contrast, overall values of k, in reverse micelles, kYiv, were always 2-4-times higher than k:. This apparent superactivity can, however, be ascribed to concentration effects.No 'bell-curve' to describe the w, dependence of catalase activity in reverse micelles was found, k or k, increasing monotonously up to wo = 50. Abbreviations. BLC, bovine liver catalase; AOT, sodium bis(2-ethylhexy1)sulfosuccinate; wnr ratio of molar concentration of water to the molar concentration of AOT (wl, = (H,O]/[AOT]); kh, k in buffer solution; k'", k in reverse micellar system; k,, the specificity constant (kcaJKM) ; k::kP, k, in reverse micelles related to water-pool concentrations; k&, k, i n reverse micelles related to the overall concentrations ; k,,, the substrate-exchange constant in the micellar system; vn, initial velocity.Enzyme. Bovine liver catalase (H,O,:H,O, oxidoreductase) (E. C. 1.11.1.6).Chemistry, Polish Academy of Sciences, 30 239 Cracow, Poland Native catalase is a tetrameric oxidoreductase with a relative molecular mass of 240000 [ 5 ] , catalyzing the conversion of water-soluble substrates such as hydrogen peroxide alone (in the catalatic activity) or together with low-chain donors such as alcohols, hydroxylamine or formic acid (in the peroxidatic activity) [6, 71. Catalase is a very fast enzyme (the catalytic constant, k,,, = 3.8 X lo7 s-', the Michaelis constant K,,, = 1.1 M and k,,,/K, = 3.5 X lo7 M-' s-' for the decomposition of H,O, at pH 7.0 [S]); this rate is thus comparable to the rate characterizing the dynamics of reversemicellar systems (the exchange rate constant, k,.., fir the intermicellar exchange of reactants is of the order 10h-108 M-' s-' [9, lo]). The effective rate constant measured for catalase in reverse micelles may therefore be dependent on the mass exchange between the micelles. If the enzymic reaction is faster than the exchange of substrate between the micelles, k,, will become the rate-limiting step. The observed rate consta...

Retracted Article: On the enzymatic activity of catalase: an iron L-edge X-ray absorption study of the active centre

“…The deprotonation of the former leads to the elongation and polarization of the O-O bond, which breaks as a peroxide oxygen, is coordinated to the Fe centre and then evolves into a Fe IV QO moiety and a haem radical. This explanation, fully based on structural arguments, needs, however, to include the lack of a noticeable temperature effect on the enzymatic activity of catalase, 14,15 which seems to be supported by our L-edge spectra and the fact that this activity is present even below the dynamic transition observed for proteins. 13 …”

“…The weak temperature effect observed here is in agreement with the almost temperature-independent activity of catalase previously reported. [13][14][15] …”

Effect of AOT on enzymatic activity of the organic solvent resistant tyrosinase from Streptomyces sp. REN-21 in aqueous solutions and water-in-oil microemulsions

“…The kinetics of very fast enzymatic reactions is determined by the dynamics of reverse micelles and water-in-oil microemulsions as it was observed for catalase in AOT/ isooctane microemulsions [6]. The kinetics of most enzymatic reactions, which are several orders of magnitude slower than the dynamics of the micellar system, may be described by other parameters of the system.…”

Influence of the hydrophobicity of lipase isoenzymes from Candida rugosa on its hydrolytic activity in reverse micelles

“…the higher interaction of isolipase A in hydrophobic chromatography [18]. Distinct emission intensities in water and in micelles but without any shift of ,~ .... have been reported for porcine pancreatic lipase [47] and bovine liver catalase [56].…”