DELETION MUTAGENESIS AS A TEST OF EVOLUTIONARY RELATEDNESS OF INDOLEGLYCEROL PHOSPHATE SYNTHASE WITH OTHER TIM BARREL ENZYMES

The role of the extra helix alpha(0) in the IV-terminal extension of t he eight-fold beta alpha barrel of indoleglycerol phosphate synthase,v as probed by point mutation and truncation, Replacing invariant leucin e 5 by valine of the enzyme from Escherichia coli affected neither k(c at) nor K-M, but deletion of 8 N-terminal residues decreased solubilit y strongly, The similarly truncated variant from the hyperthermophile Sulfolobus solfataricus was soluble, and had the same k(cat) value as the wild-type protein but a 220-fold greater K-M value, These results suggest that the N-terminal portion of helix alpha(0) provides for str ong binding of the substrate, but is not essential for stabilizing the bound transition state, Thus, three enzymes of tryptophan biosynthesi s operate essentially as canonical eight-fold beta alpha barrels, as r equired for their divergent evolution. (C) 1997 Federation of European Biochemical Societies.