Purification and Properties of the B Component of Escherichia coli Tryptophan Synthetase

Abstract

SUMMARY The procedure used to purify the B subunit of Escherichia coli tryptophan synthetase is described. The purified protein ap- pears nearly homogeneous in the ultracentrifuge and in starch gel electrophoresis. Its sedimentation coefficient was determined to be 5.0X, and a molecular weight of 108,000 was calculated from ultracentrifuge experiments. Pyridoxal phosphate has been established as the cofactor of the enzyme; approximately 2 moles are bound to each mole of B protein. B protein was re- solved into an apoenzyme which is catalytically inactive in the pyridoxal phosphate-dependent conversion of indole to trypto- phan but which retains its capacity to aid the A protein in indolyl- glycerol phosphate cleavage. Treatment with sodium borohy- dride results in a protein which is inactive in the indole to tryptophan conversion but is more active than the native protein in assisting the A subunit in the cleavage of indolylglyc- erol phosphate. A calculation based on the specific activities and molecular weights of the purified tryptophan synthetase subunits and their complex suggests that the catalytically active AB complex consists of 2 A protein molecules and 1 B protein molecule. REFERENCES