HlyC, the internal protein acyltransferase that activates hemolysin toxin:The role of conserved tyrosine and arginine residues in enzymatic activityas probed by chemical modification and site-directed mutagenesis

Internal fatty acylation of proteins is a recognized means of modifying bio logical behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of t wo lysine residue E-amino groups; HlyC catalyzes the acyl transfer from acy l-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues a mong the respective homologous C proteins that activate 13 different RTX (r epeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved t yrosines and two conserved arginines were investigated by noting the effect s of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presen ce of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutatio ns Y70F and Y150F exhibited wild-type activity. Modification of arginine re sidues with PC markedly lowered acyltransferase activity with moderate prot ection by both myristoyl-ACP and ACPSH. Under optimum conditions, four sepa rate mutations of the two conserved arginine residues (R24A, R24K, R87A, an d R87K) had little effect on acyltransferase activity.