Active-site mutations of the diphtheria toxin catalytic domain: role of 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 active-site structure and function in the ADP-ribosyltransferases. Earlier evidence suggested that histidine-21 of DT is important for the ADP-ribosylation of eukaryotic elongation factor 2 (EF-2). We have generated substitutions of this residue by cassette mutagenesis of a synthetic gene encoding the catalytic A fragment (DTA) of DT, and have characterized purified mutant forms of this domain. Changing histidine-21 to alanine, aspartic acid, leucine, glutamine, or arginine diminished ADP-ribosylation activity by 70-fold or greater. In contrast, asparagine proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by < 3-fold. The asparagine mutant was approximately 50-fold-attenuated in NAD glycohydrolase activity, however. Dissociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 microM for wild-type DTA, 160 microM for the asparagine mutant, and greater than 500 microM NAD for the alanine, leucine, glutamine, and arginine mutants. These and previous results support a model of the ADP-ribosylation of EF-2 in which histidine-21 serves primarily a hydrogen-bonding function. We propose that the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinamide 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.
Blanke
S R
SR
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts.
Huang
K
K
Wilson
B A
BA
Papini
E
E
Covacci
A
A
Collier
R J
RJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005813:Genes, Synthetic; D006639:Histidine; D006860:Hydrogen Bonding; D007700:Kinetics; D016254:Mutagenesis, Insertional; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006639:Histidine; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase