Threonine 183 and adjacent flexible loop residues in the tryptophan synthase alpha subunit have critical roles in modulating the enzymatic activities of the beta subunit in the alpha 2 beta 2 complex.

Abstract

This study investigates the catalytic and allosteric roles of a flexible loop in the tryptophan synthase alpha 2 beta 2 complex. This loop connects helix 6 and strand 6 in the alpha subunit, an 8-fold alpha/beta barrel polypeptide. We have engineered three mutations in this disordered loop: a deletion of residues 185-187 and the replacement of threonine 183 by serine (T183S) or by alanine (T183A). Position 183 is a site of an inactivating mutation identified by Yanofsky's group (Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton, B. C. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 296-298). The three engineered alpha subunits form stable, stoichiometric alpha 2 beta 2 complexes with the beta subunit which bind alpha and beta subunit ligands. Although changing threonine 183 to serine has little effect on the enzymatic properties, changing threonine 183 to alanine or deleting residues 185-187 results in a 50-fold reduction in the intrinsic activity of the alpha subunit alone and in the alpha site activity of the alpha 2 beta 2 complex. The latter two mutations profoundly alter the way in which the alpha subunit modulates the spectral properties and the activities of the wild-type beta subunit. These mutations also eliminate the effects of alpha subunit ligands on the beta subunit. Although the beta subunit ligand, L-serine, greatly stabilizes the wild-type alpha 2 beta 2 complex to dissociation and to proteolysis, L-serine stabilizes the T183A alpha 2 beta 2 complex weakly or not at all. Our findings suggest that the hydroxyl residue at position 183 and the adjacent residues in the alpha subunit loop play critical roles in the reciprocal communication between the alpha and beta subunits in the alpha 2 beta 2 complex. The results also help to explain how the wild-type alpha subunit or ammonium ion modulates the activities of the beta subunit.