IDENTIFICATION OF GLU(173) AS THE CRITICAL AMINO-ACID RESIDUE FOR THEADP-RIBOSYLTRANSFERASE ACTIVITY OF CLOSTRIDIUM-BOTULINUM C3 EXOENZYME

Clostridium botulinum C3 exoenzyme specifically ADP-ribosylates rho-p2 1 in eukaryotic cells, Trp(18) and Glu(173) of this enzyme were substi tuted with other amino acids via site-directed mutagenesis. All substi tutions at Glu(173) caused a significant reduction in affinity for NAD and diminished ADP-ribosyltransferase activity. On the other hand, th e activity of enzymes with the substitution at Trp(18) remained intact , Swiss 3T3 cells treated with the enzyme with the Trp's substitution showed the typical morphologic changes of the C3 exoenzyme phenotype, In contrast, no changes were found in cells incubated with the Glu(173 )-substituted enzyme. These results indicate that the Glu(173) residue of the C3 exoenzyme plays a key role in interacting with NAD and in e xpression of ADP-ribosyltransferase activity, which is essential for t he phenotypic change by C3 exoenzyme treatment.