Structure and Function of the Tryptophan Synthase a2b2 Complex

To probe the structural and functional roles of activesite residues in the tryptophan synthase a2b2 complex from Salmonella typhimurium, we have determined the effects of mutation of His in the b subunit. His is located adjacent to b subunit Lys, which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the a-proton of L-serine. The replacement of His by leucine (H86L) weakened pyridoxal phosphate binding ;20-fold and abolished the circular dichroism signals of the bound coenzyme and of a reaction intermediate. Correlation of these results with previous crystal structures indicates that b-His plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the b subunit. The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the synthesis of L-tryptophan from pH 7.5 to 8.8. We propose that the interaction of His with the phosphate of pyridoxal phosphate and with Lys lowers the pKa of Lys 87