THREONINE-81 OF THE TRP REPRESSOR OF ESCHERICHIA-COLI PLAYS AN AUXILIARY ROLE IN THE FORMATION OF THE COREPRESSOR BINDING POCKET

A mutational study was performed on the corepressor (L-tryptophan) bin ding site of the frp repressor of Escherichia coli. Threonine 81, one of the residues forming the hydrophobic pocket of the binding site, wa s replaced with Ser, Cys and Met by cassette mutagenesis. Biochemical characterization showed that all these mutations caused a moderate dec rease in tryptophan binding activity (free energy change similar to 1 kcal/mol). The results suggested that the binding pocket is rather fle xible in the vicinity of Thr81. On the other hand, the mutations produ ced a discernible decrease in the repressor activity in vivo, apparent ly by weakening or eliminating the hydrogen bond between Thr81 and the operator DNA, as well as by introducing possible side-chain rearrange ment.