PURIFICATION AND CHARACTERIZATION OF O-ACETYLSERINE SULFHYDRYLASE ISOENZYMES FROM DATURA-INNOXIA

Three isoenzyme forms (designated A, B, and C) of O-acetylserine sulfh ydrylase were purified from Datura innoxia suspension cultures. Isoenz yme A is the most abundant form, comprising 45-60% of the total activi ty. Isoenzymes C and B comprise 35-40% and 10-20% of the activity, res pectively. The specific activities of the purified isoenzymes are simi lar (870-893 mu mol of cysteine/min/mg of protein). Molecular masses f or isoenzymes A, B, and C, estimated by analytical size exclusion high performance Liquid chromatography, are 63, 86, and 63 kDa, respective ly. Isoenzymes A and B are homodimers; isoenzyme C is a heterodimer. S pectral analysis indicates that these isoenzymes possess a pyridoxal 5 '-phosphate cofactor that binds the O-acetylserine substrate. Binding is reversible by addition of the sulfide substrate. The O-acetylserine sulfhydrylase isoenzymes are active over a broad temperature range, w ith maximum activity between 42 and 58 degrees C. They are active only between pH 7 and 8, with optimal activity at pH 7.6. Kinetic analysis indicates these enzymes are allosterically regulated and exhibit posi tive cooperativity with respect to both substrates. They are inhibited by sulfide concentrations above 200 mu M. The kinetic analysis togeth er with the physical and spectrophotometric characteristics indicate t hat the O-acetylserine sulfhydrylase enzymes have two active sites.