| Popis: |
The trpB8 mutation of Escherichia coli causes a major conformational change within the beta subunit of tryptophan synthase. The basis of this effect is a replacement of glycine 281 by arginine within a structurally important "hinge" region. The mutant subunit, beta(B8), is catalytically active only under certain conditions, both in vivo and in vitro. Physiologically, the availability of wild type alpha subunit is the most important determinant of catalytic proficiency (Zhao, G.-P., and 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 subunit of tryptophan synthase dramatically stimulates catalysis by the beta 2(B8) mutant enzyme. However, by size exclusion high performance liquid chromatography, the stability of the alpha.beta 2(B8) complex was markedly reduced in comparison with wild type. The alpha-mediated stimulation of catalysis by the beta 2(B8) mutant enzyme was enhanced by polyethylene glycol, a volume excluder. By absorption spectroscopy, it was shown that catalysis by the beta(B8) mutant protein is blocked in at least one step after the formation of a particular Schiff base intermediate (ESII). Either the alpha subunit or ammonium ion was able to overcome this block. The microenvironment of the ESII catalytic intermediate was examined by fluorescence spectroscopy. The data are consistent with a less hydrophobic environment for ESII in the beta 2(B8) mutant protein than in the wild type protein. These lines of evidence not only 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. |
