THE NAD-GLYCOHYDROLASE ACTIVITY OF THE PERTUSSIS TOXIN S1 SUBUNIT - INVOLVEMENT OF THE CATALYTIC HIS-35 RESIDUE

Pertussis toxin is a member of ADP-ribosylating bacterial toxins that are capable of catalyzing the cleavage of the N-glycosidic bond of NAD (+) and the transfer of its ADP-ribose moiety to G proteins. The catal ytic S1 subunit of pertussis toxin uses signal transducing G proteins as acceptor substrates but can also catalyze the transfer of the ADP-r ibose moiety to water in the absence of G proteins. Site-directed muta genesis followed by kinetic analyses of truncated soluble mutant prote ins revealed that His-35 of S1 is a catalytic residue because alterati ons of this residue affect the turnover rate of NAD-glycohydrolysis by approximately two orders of magnitude without significantly affecting substrate binding. Replacement of the imidazole of His-35 by the side chain of glutamine maintained the highest residual activity. The pH d ependence of the enzyme activity showed only slight variations over th e experimental range with an optimum at pH 7.5 and an approximate pK(a ) of 6.5 to 7. This pH dependence was abolished by the Gln substitutio n, which still retained significant activity, suggesting that His-35 p robably does not act as a true base but rather as a proton acceptor. D irect catalytic roles for several other residues were ruled out. Ser-5 2 substitutions resulted in slight alterations of both K-cat and K-m f or NAD(+) suggesting an involvement in maintaining the local geometry of the active site rather than a direct role in catalysis for this res idue. Kinetic studies on mutants with substitutions of Ser-40 indicate a role in NAD(+) binding for this residue. In conjunction with previo us findings, these studies suggest that the NAD-glycohydrolase activit y of S1 utilizes 2 catalytic residues, His-35 and the previously ident ified Glu-129. The enzyme mechanism could therefore proceed through an activation by polarization of the acceptor substrate water or G prote in by His-35, and the stabilization of an oxocarbonium-like transition state intermediate by Glu-129.