The tryptophan synthase Alpha2Beta2 complex from Escherichia coli and Salmonella typhimurium is an excellent system for studying the effects of protein interaction upon protein function since the enzyme complex is readily available and is the product of a well studied genetic system. Tryptophan synthase is thus a good model for interacting proteins in more complicated biological systems. We have developed methods for purifying and crystallizing the Alpha2Beta2 complex and the separate Alpha and Beta2 subunits and for studying the two independent folding domains in each of the separate subunits. We have prepared crystals of the Alphaa2Beta2 complex from S. typhimurium which are suitable for x-ray crystallographic studies and are collaborating with crystallographers in full structural investigation of the crystalline tryptophan synthase and its complexes with substrate analogs. An electron density map is being prepared which should show the interrelationships between Alpha and Beta2 subunits and their domains and their separate active sites in the Alpha2Beta2 complex. We are also synthesizing substrate analogs which will be useful for studies using x-ray crystallography, microspectrophotometry, fluorine-NMR, rapid-scanning stopped-flow spectrophotometry, and other spectrophotometric techniques. Three of these analogs, oxindolyl-L-alanine and the two diastereoisomers of 2,3-dihydro-L-tryptophan, have been useful in probing the mechanisms of the related enzymes, tryptophanase and tryptophan synthase. Our evendence that these enzymes catalyze their similar reactions via indolenine intermediates which are stereochemically different suggests that these enzymes have different active sites and have evolved independently. 5-Flouro-L-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan are useful for fluorine-NMR studies of tryptophan synthase. 6-Azido-L-tryptophan is a reversible inhibitor of tryptophan synthase in the dark and an irreversible photoaffinity label of the Beta2 subunit in the alpha2Beta2 complex. The single essential arginine of the tryptophan synthase Alpha subunit has been identified as Arg-179 following labeling by phenylglyoxal or by NAD : arginine ADP-ribosyltransferase.
