Tryptophan Synthetase from Bacillus subtilis: PURIFICATION AND CHARACTERIZATION OF THE β2 COMPONENT

Abstract

Abstract The tryptophan synthetase β2 component of Bacillus subtilis has been purified to homogeneity using conventional techniques carried out in the presence of buffers containing 15% glycerol (v/v). Ultracentrifugal analysis of holo-β2 component indicates that it has a molecular weight of 82,000. The β chain molecular weight was estimated to be 41,000 by ultracentrifugal analysis in the presence of 5 m guanidine hydrochloride plus thiols and by sodium dodecyl sulfate polyacrylamide electrophoresis. The β chain molecular weight was also determined by Sephadex G-100 chromatography to be 41,800 using relatively mild conditions to dissociate the β2 subunit, namely 0.1 m potassium phosphate buffer, pH 7.5, containing 30% glycerol (v/v) and 0.01 m glutamine. The purified β2 subunit retains a requirement for glycerol to maintain full activity. Furthermore, contrary to earlier reports using partially purified enzyme, the β2 subunit requires the tryptophan synthetase α subunit to attain maximal activity. The B. subtilis β2 subunit appears unique among the bacterial enzymes purified thus far for its low specific activity in the reaction involving the conversion of indole plus l-serine to l-tryptophan. It is however possible for the α subunits from Escherichia coli and Pseudomonas putida to complement the β2 subunit of B. subtilis in this latter reaction and produce a level of activity equal to 30% of the homologous complementation. The B. subtilis β2 component also shares antigenic determinants with the E. coli and P. putida β2 components as well as with the tryptophan synthetase of the eucaryote, Neurospora crassa.