Tryptophan synthase: partial purification and some properties of the B-protein subunit from pea plants (Pisum sativum cv. Alaska)

Abstract

A method was developed for partial purification of the B-protein of tryptophan synthase (EC 4.2.1.20) from pea plants. The enzyme was purified 28-fold with about 21% recovery. The purification procedure removed both A-protein activity, and denatured A-protein, from the B-protein. The B-protein is unstable and activity was not preserved by either dithiothreitol, mercaptoethanol, or L-cysteine. These sulfhydryl compounds were inhibitory at relatively low concentrations. Both pyridoxal phosphate and glycerol preserved the activity to some extent. Glycerol itself was inhibitory. However, when enzyme was stored with 25% glycerol in the cold the activity actually increased within the first 24 h. The enzyme is most stable as a suspension in ammonium sulfate solution. The partially purified B-protein, in the absence of A-protein, catalyzed the condensation of indole and serine to tryptophan at an appreciable rate. Pyridoxal phosphate was required for maximal activity while both pyridoxine phosphate and pyridoxamine phosphate were inactive in the system. The molecular weight of the B-protein was estimated to be about 101 000, which is close to that of the B-protein subunit of Escherichia coli. Differences in properties between bacterial, tobacco, and pea TSase are discussed.