Structure and function of the tryptophan synthase alpha(2)beta(2) complex - Roles of beta subunit histidine 86

To probe the structural and functional roles of active-site residues in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium, w e have determined the effects of mutation of His(86) in the beta subunit. H is(86) is located adjacent to beta subunit Lys(87), which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the alpha-proton of L-serine. The replacement of His(86) by leucine (H86L) wea kened pyridoxal phosphate binding similar to 20-fold and abolished the circ ular dichroism signals of the bound coenzyme and of a reaction intermediate . Correlation of these results with previous crystal structures indicates t hat beta-His(86) plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the activ e site of the beta subunit. The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the s ynthesis of L-tryptophan from pH 7.5 to 8.8. We propose that the interactio n of His(86) with the phosphate of pyridoxal phosphate and with Lys(87) low ers the pK(a) of Lys(87) in the wild-type alpha(2)beta(2) complex and there by facilitates catalysis by Lys(87) in the physiological pH range.