Abstract
O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55% by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25% and 12% by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21%. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the β-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40°C (when tested at pH 7.5), and at pH 6.5 (at 40°C). The enzyme kept 90% its activity after incubation at 65°C (at pH 7.5) for 30 min, and more than 90% after 30 min incubation at pHs 7-12 at 30°C. The enzyme had a Km of 4 mM for O-acetyl-L-serine and a Vmax of 37.0 μmol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5% total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5′-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.
