d‐Galactose Dehydrogenase from Pseudomonas saccharophila

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Abstract
D‐Galactose dehydrogenase from Pseudomonas saccharophila has been purified over 700‐fold with an over‐all yield of 27%. The purified enzyme exhibits a specific activity of about 200 μmol NADH formed × min−1× mg protein−1 and has been shown to be homogeneous by ultracentrifugation and by gel electrophoresis. The amino acid composition of the enzyme has been determined. The native enzyme has a sedimentation coefficient (S20,w0) of 5.94 S and a diffusion coefficient (D20,w0) of 5.60 F. The experimentally determined partial specific volume (v̄) is 0.744 ml/g. The molecular weight calculated from these data is 103000, in good agreement with the value of 102000 estimated by gel permeation chromatography. The enzyme dissociates into polypeptide chains in the presence of 5 M guanidine hydrochloride or 0.1% sodium dodecylsulfate. The molecular weight of the polypeptide chains has been found to be 25000 by ultracentrifugation (S20,w0= 1.35 S, D20,w0= 5.28 F) and by dodecylsulfate‐polycrylamide electrophoresis. The native enzyme is composed of four polypeptide chains. The pI of d‐galactose dehydrogenase is 5.1. The pH‐optimum is between pH 8–9 in Tris‐HCI buffer. AT PH 8.6 and 30°C the limiting michaelis constant for NAD+ is Ka= 0.14 mM, the limiting michaelis constant for d‐galactose is Kb= 1.0 mM and the dissociation constant for NAD+ is Kia= 0.48 mM. The enzyme is specific for NAD and uses NADP only to a minor extent as a coenzyme. It oxidizes the following sugars arranged according to relative rates, in decreasing order: d‐FUCOSE > d‐galactose > 3‐deoxy‐d‐galactose > 2‐deoxy‐d‐galactose > 4‐deoxy‐d‐galactose > l‐arabinose > 2‐deoxy‐2‐amino‐d‐galactose.