Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.2.42 (DTA)
 1,693 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have suggested that tyrosine-65 (Tyr-65) of diphtheria toxin (DT) is located at the active site. To investigate the role of Tyr-65 in NAD binding and the ADP-ribosylation of elongation factor-2 (EF-2), we changed this residue to alanine and phenylalanine by site-directed mutagenesis of a synthetic gene encoding the catalytic fragment of DT (DTA). The alanine mutant was greatly diminished in ADP-ribosylation activity (350-fold) and NAD-glycohydrolase activity (88-fold), whereas the phenylalanine mutant was reduced in these activities only slightly. Dissociation constants (Kd) for NAD binding were 15 microM for wild-type DTA, 26 microM for the phenylalanine mutant, and greater than 800 microM NAD for the alanine mutant. However, both mutant enzymes were found to bind adenosine with nearly equal affinity as wild-type DTA. These results support a model of ADP-ribosylation in which the phenolic ring of Tyr-65 interacts with the nicotinamide ring of NAD, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism.
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PMID:Active-site mutations of diphtheria toxin: role of tyrosine-65 in NAD binding and ADP-ribosylation. 780 11

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.
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PMID: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. 817 90