Abstract
A new enzyme, N-acetyl-D-hexosamine dehydrogenase (N-acetyl-α-D-hexosamine:NAD+ 1-oxidoreductase), was purified to homogeneity on polyacrylamide gel electrophoresis from a strain of Pseudomonas sp. about 900-fold with a yield of 12%. The molecular weight of the enzyme was about 124, 000 on gel filtration and 30, 000 on SDS-polyacrylamide gel electrophoresis, respectively. Its isoelectric point was 4.7. The optimum pH was about 10.0. The enzyme was most stable between pH 8.0 and pH 10.5. The highest enzyme activity was observed with N-acetyl-D-glucosamine (Km=5.3mM) and N-acetyl-D-galactosamine (Km=0.8mM) as the sugar substrate. But it was not so active on N-acetyl-D-mannosamine. NAD+ was used specifically as the hydrogen acceptor. The anomeric requirement of the enzyme for N-acetyl-D-glucosamine was the α-pyranose form, and the reaction product was N-acetyl-D-glucosaminic acid. The enzyme activity was inhibited by Hg2+ and SDS, but many divalent cations, metal-chela ting reagents, and sulfhydryl reagents had no effect.
