THREONINE-74 IS A KEY SITE FOR THE ACTIVITY OF CLOSTRIDIUM-PERFRINGENS ALPHA-TOXIN

A mutant toxin (MT) that abolished almost 99% of the hemolytic activit y of alpha-toxin was isolated by random polymerase chain reaction (PCR ) mutagenesis of the gene for Clostridium perfringens alphatoxin. In t he mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I) -345 were substituted with His, Ile and Met, respectively, Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-f old of that of the wild-type, The replacement of Y-62 with Ile or I-34 5 with Met alone did not affect the activities of the toxin. T74I muta nt bound to sheep erythrocyte membranes and specifically bound [Zn-65] (2+) in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein, These results sugges t that the T-74 residue plays a key role in these biological activitie s of C. perfringens alpha-toxin.