Structure and Function of the Tryptophan Synthase α2β2 Complex

Abstract

To probe the structural and functional roles of active-site residues in the tryptophan synthase α2β2 complex from Salmonella typhimurium, we have determined the effects of mutation of His86 in the β subunit. His86 is located adjacent to β subunit Lys87, which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the α-proton of L- serine. The replacement of His86 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 β-His86 plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the β 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 His86 with the phosphate of pyridoxal phosphate and with Lys87 lowers the pK(a) of Lys87 in the wild- type α2β2 complex and thereby facilitates catalysis by Lys87 in the physiological pH range.