HlyC, the internal protein acyltransferase that activates hemolysin toxin:Roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis

Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protei n is the essential acyl donor. The acyltransferase reaction progresses thro ugh formation of a binary complex between acyl-ACP and HlyC to a reactive a cyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of t he family have a number of conserved amino acid residues that may be cataly tically important. Experiments to illuminate the reaction mechanism were do ne. The formation of an acyl-enzyme intermediate suggested that the reactio n likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, a nd expressed substrates and enzyme. The-effects of single site-directed mut ations of conserved residues of HlyC on different portions of reaction prog ress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the onl y residue essential for activity, formed normal binary complexes but were u nable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glyci nes results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-typ e.