IMPORTANCE OF CONSERVED AND VARIABLE C-TERMINAL RESIDUES FOR THE ACTIVITY AND THERMAL-STABILITY OF THE BETA-SUBUNIT OF TRYPTOPHAN SYNTHASE

To assess the functional roles of helix 13 and of the conserved and va riable residues in the C-terminal region (residues 378-397) of the try ptophan synthase beta subunit, we have constructed four C-terminal tru ncations and 12 point mutations. The effects of these mutations on kin etic and spectroscopic properties and thermal stability are reported h ere. The mutant beta subunits all form stable alpha(2) beta(2) complex es that have been purified to homogeneity. The mutant alpha(2) beta(2) complexes are divided into two classes on the basis of activity in th e reaction of L-serine with indole to form tryptophan. Class I enzymes , which have mutations at Arg-379 or Asp-381 or truncations (384-397 o r 385-397), exhibit significant activity (138% of wild type). Class II enzymes, which have mutations at Lys-382 or Asp 383 or truncations (3 82-397 or 385-397), exhibit very low activity (<1% of wild type), Alth ough Class II enzymes have drastically reduced activity in the reactio n of L-serine with indole and an altered distribution of enzyme-substr ate intermediates in the reaction of L-serine with beta-mercaptoethano l, they retain activity in the reaction of beta-chloro-L-alanine with indole. Correlation of the results with the three-dimensional structur e of the alpha(2) beta(2) complex suggests that Lys-382 and Asp-383 se rve important roles in a proposed ''open'' to ''closed'' conformationa l change that occurs in the reactions of L-serine, Because mutant beta subunits having C-terminal truncations (383-397 or 384-397) undergo m uch more rapid thermal inactivation at 60 degrees C than the wild type beta subunit, the C-terminal helix 13 stabilizes the beta subunit.