Activation of hemolysin toxin: relationship between two internal protein sites of acylation

HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA). The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites. The substrate specificity of acylation was examined. ProHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site-directed mutation analyses of acylation sites, often served as internal protein acylation substrates, and the kinetics of the acylations were measured. The two sites of acylation of proHlyA functioned independently of one another with HlyC; there did not appear to be a common HlyC binding site or processivity of the enzyme between the sites. Acyl-HlyC was likely the enzyme form that interacted with the final acylation substrate. In a variety of constructs, the two acylation sites had similar K(m) values, but their V(max) values and catalytic efficiencies as substrates differed. Internal protein acylation was inhibited by specific small peptides mimicking the primary structure of each acylation site except that the crucial lysines were replaced with arginines; similar small peptides containing the crucial lysine, however, were not acylated.