The B Protein of Escherichia coli Tryptophan Synthetase: I. EFFECTS OF SULFHYDRYL MODIFICATION ON ENZYMATIC ACTIVITIES AND SUBUNIT INTERACTION

Abstract

Abstract Sulfhydryl modification of the B protein of Escherichia coli tryptophan synthetase has been carried out to investigate the role of sulfhydryl residues in the active center of this protein and in its interaction with the A protein. Both N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with two sulfhydryls per monomer of apo-B protein and one sulfhydryl per monomer of the holo-B protein, which contains 1 mole of pyridoxal phosphate per monomer. The serine deaminase activity and the tryptophan synthetase activity are both largely lost after the reaction of one sulfhydryl in the holo-B protein with DTNB. Similar results are obtained after the reaction of one sulfhydryl in the apo-B protein with NEM. In contrast, after the reaction of one sulfhydryl in the holo-B protein with NEM, the serine deaminase activity is somewhat enhanced, whereas, the tryptophan synthetase activity is largely lost. Kinetic comparisons of the NEM-modified holo-B protein with the untreated holo-B protein show that the Km for l-serine is largely unchanged, but that both the Km for indole and the Vmax in the tryptophan synthetase reaction are changed by modification. Since one sulfhydryl in the B protein is fully protected from NEM modification by pyridoxal phosphate and a second sulfhydryl is partially protected from modification in the holo-B protein by the substrate l-serine, both of these sulfhydryl residues may be located in the active center of the protein. The reactive sulfhydryl residue in the holo-B protein is also protected by interaction with the A protein. However, this sulfhydryl is not necessary for the interaction of the A and B proteins since the holo-B protein modified with 1 mole of NEM still associates normally with the A protein and facilitates its hydrolysis of indole-3-glycerol phosphate.