Mirella Pilone Simonetta

Induction of d-Amino-acid Oxidase by d-Alanine in Rhodotorula gracilis Grown in Defined Medium

SUMMARY: The obligate aerobe yeast Rhodotorula gracilis was grown in batch culture on a chemically defined, pH-controlled medium containing glucose or d-alanine as carbon sources, ammonium or d-alanine as nitrogen sources, and d-alanine as a sole carbon and nitrogen source. Under these conditions, d-alanine induced the synthesis of d-amino-acid oxidase (EC 1.4.3.3) to an extent depending on the nutrients, the highest specific activity of the enzyme [up to 0.6 U (mg protein)−1] being detected when both d-alanine and glucose were present in the growth medium. In contrast, enzyme activity was negligible when both ammonium and glucose were present in the growth medium, even in the presence of d-alanine. The racemic mixture dl-alanine was also utilized as a source of both carbon and nitrogen for the growth of R. gracilis, but the enzyme activity appeared only after the depletion of l-alanine from the medium. Data on transmembrane transport of d-alanine in the presence of different nutrients clearly indicated that the l-isomer prevented induction by d-alanine through inhibition of the transport of the d-amino acid into cells. However, such an effect was not exerted by ammonium, indicating that this compound probably acts at the level of enzyme synthesis.

Purification and properties of d-amino-acid oxidase, an inducible flavoenzyme from Rhodotorula gracilis☆

Abstract d -Amino-acid oxidase ( d -amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) was purified about 950-fold from the red yeast Rhodotorula gracilis . The procedure gave an enzyme preparation which is greater than 90% homogeneous on SDS-polyacrylamide gels with a specific activity of 58 U/mg at 37°C with d -alanine as substrate. d -Amino-acid oxidase from yeast is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The subunit molecular weight is 37000, while the native enzyme is a dimer of 79000 as determined by SDS-polyacrylamide gel electrophoresis and gel-filtration chromatography, respectively. The enzyme from Rhodotorula oxidizes several d -amino acids and activity was also detected on thiazolidine-2-carboxylic acid. Substrate specificity and inhibition by benzoate differ from the ones exhibited by the mammalian enzyme and the recently identified d -amino-acid oxidase from Trigonopsis variabilis .

Kinetic changes following modification of rat liver alcohol dehydrogenase by deoxycholate

Abstract Deoxycholate and other bile steroids activate rat liver alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1). The kinetic changes following the enzyme modification by deoxycholate were studied in both directions of the reaction ethanol + NAD+ ⇌ acetaldehyde + NADH using a purified enzyme preparation. Initial rate measurements and analysis of product inhibition patterns show a particularly significant increase of the Michaelis and inhibition constants for ethanol and acetaldehyde, and also an overall change of the reaction mechanism. The kinetic pattern of the unmodified enzyme is consistent with a mechanism of the Theorell-Chance type, with kinetically irrelevant ternary complexes. The modification by 1 m m deoxycholate causes a transition toward a quite different reaction sequence. Data are inconsistent with a simple ordered mechanism and make evident the existence of a more complicated mechanism, which may include conformational changes of the binary complexes and partial randomization.

MIR: A versatile program for the statistical analysis of enzyme kinetic data

We have developed a package program for the estimation of Michaelis-Menten parameters for enzymes that conform to different kinetic mechanisms. Data from different experimental schemes can be fitted with appropriate weighing factors to any of 6 mathematical models, corresponding to 5 kinetic mechanisms: ordered bi-bi, Theorell-Chance, rapid equilibrium random bi-bi, rapid equilibrium ordered bi-bi and ping pong bi-bi. The program also performs a significance test to discriminate between different candidate models. To illustrate the performance of the program, real data from kinetic experiments with glucose 6-phosphate from Leuconostoc mesenteroides have been fitted to different mathematical models, and the results are discussed. The program can be easily implemented for the fitting of kinetic data to any other model.

Regulation of enzymes of ethanol metabolism in yeast (Rhodotorula gracilis)

The three enzymes of ethanol metabolism alcohol dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase in the obligate aerobic yeastRhodotorula gracilis are repressed by glucose and induced by C2 metabolic fuels with a regulatory pattern indicating a correlation in the control mechanisms. To try an identification of the molecular signals involved in the transmission of the inducing stimulus, experiments were carried out by blocking with 2 mM pyrazole the ethanol ↼ ↼ acetaldehyde metabolic step. Results indicate that ethanol is not specifically required as a molecular signal for induction.

The effect of pH and methylation on the interaction of deoxycholate with rat liver alcohol dehydrogenase

The activation of rat liver alcohol dehydrogenase by deoxycholate depends on the anionic form of the steroid. Methylation of the enzyme protein leads to an increase of both turnover number and Km for ethanol and to a change in the effect of deoxycholate, which behaves as an inhibitor. It is suggested that the steroid and methylation effects depend on the same basic mechanism, in which one or more Lys groups are involved.