1. A procedure is described for the isolation of D-galactose dehydrogenase from Pseudomonas fluorescens, grown on D-galactose as carbon source. A 750-fold purification was achieved with an overall yield of about 40%. The final preparation had a specific activity of about 850 pmo1 NADH formed per min per mg protein.2. The enzyme has been shown to be homogeneous by ultracentrifugation, by disc gel electrophoresis, by gel isoelectric focusing and by dodecylsulfate gel electrophoresis.3. The molecular weight of the native enzyme has been determined to be 64000 by sedimentation equilibrium studies and by gel permeation chromatography.4. The enzyme dissociates in the presence of 0.1 % sodium dodecylsulfate to polypeptide chains, whose molecular weight was determined as 32000 by electrophoresis, indicating that the native enzyme is composed of two subunits.5. The amino acid composition of D-galactose dehydrogenase has been determined. 6. The PI of the enzyme has been shown to be 4.28. The pH optimum is between 9.1 and 9.5. At pH 9.1 and 30 "C the limiting Michaelis constant for NAD+ is Ka = 0.24 mM, for NADP+ Ka = 2.3 mM and for D-galactose with NAD+ and with NADP+ as cosubstrate Kb = 0.7 mM. The dissociat on constant for NAD+ is Kia = 0.54 mM and for NADPI-Kia = 6.2 mM. 8. The correspondence between amino acid composition, subunit size and structure and between specificity of D-galactose dehydrogenase from Ps. fluorescens and from Ps. saccharophila is discussed.

1. b-D-Galactopyranose has been shown by specificity studies and by gas-liquid chromatographic investigations to be the substrate of the D-galactose dehydrogenases from Pseudomonas sacchurophilu and from Pseudomonas fluorescens.2. cc-~-Galactopyranose and the two ring-isomeric D-galactofuranoses are oxidized, at high enzyme concentrations, only after their rate-limiting isomerization into the b-D-pyranose. The open-chained aldehyde form of D-galactose has been likewise excluded as substrate.3. D-Galactono-l,5-lactone has been shown by gas-liquid chromatography to be the immediate product of the enzymatic dehydrogenation of D-galactose and identified by combined gas-liquid chromatography -mass spectrometry of the isolated trimethylsilylated derivative.4. The primarily formed D-galactono-l,5-lactone rearranges non-enzymatically to the corresponding D-galactono-l,4-lactone by an intramolecular rearrangement without the participation of the open-chained D-galactonate. This rearrangement is strongly dependent on the pH-value, having a first-order rate constant k = 1.0 min-' at pH 6.8 under the conditions used. were determined at pH 6.8 under the conditions used, enabling the estimation of the apparent equilibrium constant for the reaction catalyzed by the enzyme6. Results analogous to those with D-galactose were obtained from investigations with the homeomorphic L-arabinose. The pyranose ring with the hydroxyl group at carbon-1 in the equatorial position is likewise the only substrate, and the immediate product, the 1,5-lactone, also rearranges subsequently to the corresponding 1,4-lactone, having a first-order rate constant k = 0.22 min-l at pH 6.8 under the conditions used. The ~-arabinono-1,5-lactone has also been identified by gas-liquid chromatography -mass spectrometry of the trimethylsilylated derivative.

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