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 converted 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 intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1999) Biochemistry 38, 9541-9548]. The ping pong kinetic mechanism implied by these findings was validated using two different acyl-ACP substrates, thus verifying the independence of the previously demonstrated two partial reactions. Assessments 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 conserved 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 significant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA. These findings are compatible with transient formation of acyl-His23 during the course of HlyC catalysis. The effects of several other single site-directed mutations of conserved residues of HlyC on different portions of the reaction progress were examined using a 39 500 kDa fragment of proHlyA which was a more effective substrate than intact proHlyA.
Worsham
L M
LM
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, Box 70 581, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Trent
M S
MS
Earls
L
L
Jolly
C
C
Ernst-Fonberg
M L
ML
D000215:Acylation; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism
D000596:Amino Acids; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D000217:Acyltransferases; C092237:HlyC protein, E coli