Yuzuru Suzuki

Enzymatic pyrophosphorylation of 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine by cell-free extracts of Escherichia coli B

Abstract Cell-free extracts of Escherichia coli B catalyze the direct pyrophosphorylation of 14C-labelled 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine. Radioisotopic and chromatographic evidence show that the product enzymatically formed in this reaction is the pyrophosphate ester of the pteridine. ATP and Mg++ are essential as cofactors for the reaction. The pyrophosphorylation is strongly inhibited by 2-amino-4-hydroxy-6-carboxy-7,8-dihydropteridine.

[158] Assay methods, isolation procedures, and catalytic properties of riboflavin synthetase from spinach

Publisher Summary This chapter discusses the assay methods, isolation procedures, and catalytic properties of riboflavin synthetase from spinach. The conversion catalyzed by riboflavin synthetase, four carbon atoms from the methyl group substituents and carbons 6 and 7 of one molecule of 6, 7-dimethyl–8-ribityllumazine are transferred to a second molecule of the lumazine to form the o-xylene ring of riboflavin. This mechanism is established both by the stoichiometry of the reaction and by the isotopic studies. Riboflavin synthetase activity is detected in extracts of a number of plants and flavinogenic microorganisms and is found in homogenates of some animal livers. Partial purification of the enzyme is performed with spinach, Escherichia coli , and Ashbya gossypii . 6, 7-Dimethyl-8-ribityllumazine is mixed with the enzyme and the riboflavin produced is measured spectrophotometrically by reading absorbancies at 470 and 405 nm, or fluorophotometrically after isolation by paper chromatography. The purification procedure for spinach enzyme is carried in seven different steps, namely––extraction, first and second ammonium sulfate fractionation, protamine sulfate treatment, CM-cellulose treatment, and first and second diethylaminoethyl (DEAE)-cellulose column chromatography.

NAD+-dependent formation of 2-amino-4-hydroxy-6-carboxypteridine from 2-amino-4-hydroxy-6-formylpteridine by cell-free extracts of Escherichia coli

Abstract Cell-free extracts of Escherichia coli B catalyze the production of 2-amino-4-hydroxy-6-carboxypteridine from 2-amino-4-hydroxy-6-formylpteridine. NAD+ is essential as a cofactor for this reaction. The product is isolated by column chromatography on Sephadex G-25 and by paper partition chromatography. The structure of the product is determined on the basis of ultraviolet spectra, behavior on paper chromatography, and pH-fluorescence curves.

Intracellular accumulation of S-adenosylmethionine in a high-riboflavinogenic Eremothecium ashbyii grown in the presence of 3-amino-1,2,4-triazole

Abstract S -Adenosylmethionine accumulated in a high-riboflavinogenic Eremothecium ashbyii when grown in the presence of 3-amino-1,2,4-triazole, a specific inhibitor of riboflavin synthesis. The accumulated substance was extracted from the mycelia with HClO 4 , and purified by the column chromatography on Dowex 1 × 2 and 50W × 8. Its structure was determined on the basis of ultraviolet spectra, behavior on paper chromatography, and hydrolysis experiments.

A second-order kinetic reaction catalyzed by riboflavin synthetase from a high-riboflavinogenic Eremothecium ashbyii

Abstract The initial reaction velocity of the reaction catalyzed by riboflavin synthetase from a high-riboflavinogenic Eremothecium ashbyii shows a second-order dependency on 6,7-dimethyl-8-ribityllumazine concentration in the range of 1.5·10 −4 –6.25·10 −6 M, suggesting that binding of the two molecules of the lumazine to the enzyme occurs with affinities of similar orders of magnitude. Binding of one lumazine molecule to the enzyme increases the affinity of the second molecule for the enzyme.