ACTIVE-SITE MUTATIONS OF THE DIPHTHERIA-TOXIN CATALYTIC DOMAIN - ROLEOF HISTIDINE-21 IN NICOTINAMIDE ADENINE-DINUCLEOTIDE BINDING AND ADP-RIBOSYLATION OF ELONGATION-FACTOR-2

Diphtheria toxin (DT) has been studied as a model for understanding ac tive-site structure and function in the ADP-ribosyltransferases. Earli er evidence suggested that histidine-21 of DT is important for the ADP -ribosylation of eukaryotic elongation factor 2 (EF-2). We have genera ted substitutions of this residue by cassette mutagenesis of a synthet ic gene encoding the catalytic A fragment (DTA) of DT, and have charac terized purified mutant. forms of this domain. Changing histidine-21 t o alanine, aspartic acid, leucine, glutamine, or arginine diminished A DP-ribosylation activity by 70-fold or greater. In contrast, asparagin e proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by <3-ford. The asparagine mutant was appro ximately 50-fold-attenuated in NAD glycohydrolase activity, however. D issociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 mu M for wild-type DTA, 1 60 mu M for the asparagine mutant, and greater than 500 mu M NAD for t he alanine, leucine, glutamine, and arginine mutants. These and previo us results support a model of the ADP-ribosylation of EF-2 in which hi stidine-21 serves primarily a hydrogen-bonding function. We propose th at the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinam ide carboxamide, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism, and then stabilizing the transition-state intermediate of this reaction.