A. Stephen Dahms

[49] d-xylo-aldonate dehydratase

Publisher Summary This chapter describes the purification and properties of the enzyme D-xylo-aldonate dehydratase that functions in the metabolism of D-xylose in the pseudomonad designated “MSU-1.” The keto acid formed during the reaction is measured as its semicarbazone by the method of MacGee and Doudoroff. The enzyme is purified from pseudomonad MSU-I. The purification procedure involves: protamine sulfate treatment, ammonium sulfate fractionation, chromatography on Sephadex G-200, and heat step. The substrate specificity of the enzyme shows that among 18 aldonic, uronic, and aldaric acids tested, only D-xylonate and D-gluconate serve as substrates. The enzyme exhibits an absolute requirement for a divalent cation. The enzyme is stable in the frozen state for several months and is not affected by thiols, iodoacetic acid, p -mercuribenzoate, or N -ethylmaleimide at 2 mM. The dehydratase is induced by growth on D-xylose, but not by D-gluconate, D-glucose, L-arabinose, or D-fucose. Although the enzyme exhibits D-gluconate dehydratase activity, it is not induced by growth on glucose or gluconate.

[42] 2-Keto-3-deoxy-d-xylonate aldolase (3-deoxy-d-pentulosonic acid aldolase)

Publisher Summary This chapter describes the purification and properties of the enzyme 2-Keto-3-deoxy-D-xylonate aldolase that functions in the metabolism of D-xylose, and, presumably, other D-pentoses in a pseudomonad designated “MSU-I.” The continuous spectrophotometric assay measures the rate of pyruvate formation by coupling the reaction to lactate dehydrogenase. With the coupling enzyme in excess, the rate of 2-keto-3-deoxy-D-xylonate cleavage is equal to the rate of NADH oxidation as monitored at 340 nm. The purification process involves protamine sulfate treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-200. Aldolase activity as a function of pH is maximal in the pH range of 7.4–8.2. The aldolase requires a divalent cation for activity when assayed in the presence of EDTA. The aldolase is insensitive to borohydride reduction, whether in the presence of pyruvate, glycolaldehyde, or 2-keto-3-deoxy-D-xylonate. The aldolases are specific for their respective enantiomeric 3-deoxypentulosonic acid substrates, have similar pH optima and equilibrium constants, and are unaffected by borohydride in the presence of either substrate or individual cleavage products.

[34] d-Aldohexose dehydrogenase

Publisher Summary This chapter describes the assay method, purification procedure, and the properties of D-aldohexose dehydrogenase. This dehydrogenase also catalyzes the oxidation of other D-aldohexoses, and apparently functions in the metabolism of D-fucose and D-glucose in pseudomonad MSU-1. The rate of D-glucose-dependent NADH formation is determined by measuring the rate of absorbance increase at 340 nm. The following are added to a microcuvette with a 1.0 cm light path: 0.05 ml of buffer, 0.01 ml of D-glucose, 0.01 ml of NAD + , D-aldohexose dehydrogenase, and water to a volume of 0.15 ml. The reaction is initiated by the addition of D-aldohexose dehydrogenase. The reaction rates are conveniently measured with a Gilford multiple-sample absorbance recorder. The cuvette compartment should be thermostated at 25°. The purification procedure includes steps such as: growth of organism, preparation of cell extracts, protamine sulfate treatment, ammonium sulfate fractionation, sephadex G-200 chromatography, DEAE-cellulose chromatography, and calcium phosphate gel step. This enzyme is specific for D-aldohexoses, the following of which serve as substrates, in order of decreasing activity: D-glucose, D-galactose, D-mannose, 2-deoxy-D-glucose, 6-deoxy-D-galacrose (D-fucose), 2-deoxy-D-galactose, D-altrose, 6-deoxy-D-glucose (Dquinivose), D-allose, and 3,6-dideoxy-D-galactose (abequose).

[47] d-Galactonate dehydratase

Publisher Summary This chapter describes the purification and properties of the enzyme galactonate dehydratase that functions in the metabolism of D-galactose and D-galactonic acid in a pseudomonad designated “MSU-1.” The α-keto acid formed during the reaction is measured as its semicarbazone by the procedure of MacGee and Doudoroff. The purification process of the enzyme involves growth of organism, preparation of cell extracts, protamine sulfate treatment, ammonium sulfate fractionation, Sephadex G-200, calcium phosphate gel, and purification by diethylaminoethyl (DEAE)-cellulose. Studies indicate that the enzyme is specific for D-galactonate, and the optimal pH is 7.1 in glycylglycine buffer. The enzyme is quite stable to thermal treatment and possesses a half-life of over 5 minutes at 90 ° C. The native protein has a molecular weight of 240,000 to 245,000 as shown by gel filtration and sedimentation on sucrose density gradients. The enzyme does not possess 2-keto-3-deoxygalactonate aldolase, 2-ketodeoxygluconate aldolase, 2-keto-3-deoxygalactonate kinase, or 2-keto-3-deoxy-6-phosphogalactonate aldolase activity.