TY - JOUR
T1 - Active-Site Mutations of the Diphtheria Toxin Catalytic Domain
T2 - Role of Histidine-21 in Nicotinamide Adenine Dinucleotide Binding and ADP-Ribosylation of Elongation Factor 2
AU - Blanke, Steven R.
AU - Huang, Kathy
AU - Wilson, Brenda A.
AU - Collier, R. John
AU - Wilson, Brenda A.
AU - Papini, Emanuele
AU - Covacci, Antonello
PY - 1994/5/1
Y1 - 1994/5/1
N2 - 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 µ? for wild-type DTA, 160 µ? for the asparagine mutant, and greater than 500 µ? 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 π-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.
AB - 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 µ? for wild-type DTA, 160 µ? for the asparagine mutant, and greater than 500 µ? 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 π-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.
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U2 - 10.1021/bi00183a019
DO - 10.1021/bi00183a019
M3 - Article
C2 - 8172890
AN - SCOPUS:0028260470
SN - 0006-2960
VL - 33
SP - 5155
EP - 5161
JO - Biochemistry
JF - Biochemistry
IS - 17
ER -