We are investigating the structure and function of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of x-ray crystallography, site-directed mutagenesis, and spectroscopic studies. The tryptophan synthase multienzyme complex catalyzes the final reaction in L-tryptophan biosynthesis and has been the subject of many genetic and biochemical studies. Our recent determination of the three-dimensional structure of this multienzyme complex by x-ray crystallography allows us to locate the active sites of both the alpha and beta subunits and a tunnel which connects these two sites. The 25 A tunnel facilitates the diffusion of indole produced at the active site of the alpha subunit to the active site of the beta subunit. Site-directed mutagenesis of tryptophan synthase has been initiated in order to investigate the effects of structure on the functional properties of tryptophan synthase. We have designed and prepared a series of mutant forms of the alpha and beta subunit by site-directed mutagenesis. More than 30 such mutant proteins have been isolated by a new efficient purification procedure in which the alpha 2 beta 2 complex is crystallized from a crude extract. Two mutants in which either cysteine-81 or cysteine-118 of the alpha subunit is replaced by a serine residue are potentially useful for x-ray crystallographic studies, since a heavy metal binding site is specifically eliminated in each mutant. Studies of four mutants in which aspartic acid-60 of the alpha subunit was changed to four different amino acids indicate that aspartic acid-60 is an essential catalytic base. Studies of single and double mutants at tyrosine-175 and cysteine-211 of the alpha subunit support a substrate binding role for these residues. Studies of mutations at several sites in the beta 2 subunit are defining the active site roles of these residues. We are also trying to produce mutations which block the tunnel which extends between the active sites of the alpha and beta subunits. Spectroscopic methods have been used to study transient reaction intermediates with substrate analogs and to study the kinetics of folding and unfolding of the alpha subunit.
