Insights into the catalytic mechanism of HlyC, the internal protein acyltransferase that activates Escherichia coli hemolysin toxin

Hemolysin, a toxic protein secreted by pathogenic Escherichia coli, is conv erted from nontoxic prohemolysin, proHlyA, to toxic hemolysin, HlyA, by an internal protein acyltransferase, HlyC. Acyl-acyl carrier protein (ACP) is the essential acyl donor. The acyltransferase reaction proceeds through two partial reactions and entails formation of a reactive acyl-HlyC intermedia te [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1999) Biochemis try 38, 9541-9548]. The ping pong kinetic mechanism implied by these findin gs was validated using two different acyl-ACP substrates, thus verifying th e independence of the previously demonstrated two partial reactions. Assess ments of the stability of the acyl-HlyC intermediate revealed an increased stability at pH 8.6 compared to more acidic pHs. Mutations of a single cons erved histidine residue essential for catalysis gave minimal activity when substituted with a tyrosine residue and no activity with a lysine residue. Unlike numerous other His23 mutants, however, the H23K enzyme showed signif icant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA. These findings are c ompatible with transient formation of acyl-His23 during the course of HlyC catalysis. The effects of several other single site-directed mutations of c onserved residues of HlyC on different portions of the reaction progress we re examined using a 39 500 kDa fragment of proHlyA which was a more effecti ve substrate than intact proHlyA.