A SINGLE AMINO-ACID SWITCH WITHIN THE HINGE REGION OF THE TRYPTOPHAN SYNTHASE BETA-SUBUNIT OF ESCHERICHIA-COLI THAT LEADS TO DIMINISHED ASSOCIATION WITH ALPHA-SUBUNIT AND ARRESTED CONVERSION OF ESII TO PRODUCT

The trpB8 mutation of Escherichia coli causes a major conformational c hange within the beta subunit of tryptophan synthase. The basis of thi s effect is a replacement of glycine 281 by arginine within a structur ally important ''hinge'' region. The mutant subunit, beta(B8), is cata lytically active only under certain conditions, both in vivo and in vi tro. Physiologically, the availability of wild type alpha subunit is t he most important determinant of catalytic proficiency (Zhao, G.-P., a nd Somerville, R. L. (1992) J. Biol. Chem. 267, 526-541; Zhao, G.-P., and Somerville, R. L. (1993) J. Biol. Chem. 268, 14912-14920). Through enzyme activity titration experiments it was shown that the alpha sub unit of tryptophan synthase dramatically stimulates catalysis by the b eta2(B8) mutant enzyme. However, by size exclusion high performance li quid chromatography, the stability of the alpha.beta2(B8) complex was markedly reduced in comparison with wild type. The alpha-mediated stim ulation of catalysis by the beta2(B8) mutant enzyme was enhanced by po lyethylene glycol, a volume excluder. By absorption spectroscopy, it w as shown that catalysis by the beta(B8) mutant protein is blocked in a t least one step after the formation of a particular Schiff base inter mediate (ESII). Either the alpha subunit or ammonium ion was able to o vercome this block. The microenvironment of the ESII catalytic interme diate was examined by fluorescence spectroscopy. The data are consiste nt with a less hydrophobic environment for ESII in the beta2(B8) mutan t protein than in the wild type protein. These lines of evidence not o nly support a conformational switch model of open versus closed states within the beta subunit during the catalytic cycle but also suggest a functional role for the hinge region in the process of conformational switching.