Active-Site Mutations of Diphtheria Toxin: Effects of Replacing Glutamic Acid-148 with Aspartic Acid, Glutamine, or Serine

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu → Asp (E148D), Glu → Gln (E148Q), and Glu → Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pK_a of 6.2-6.3 and is in the protonated state in the rate-determining step. As this titration is absent in the pH profiles of either of two mutants examined, DTA-E148Q or DTA-E148S, the apparent pK_a of 6.2-6.3 in the wild-type toxin may be due to an active-site imidazole, whose titration is affected by the presence or absence of the carboxyl group. Independent evidence indicates that there is a single histidine (His-21) within the active-site pocket of DTA, and we suggest that both His-21 and Glu-148 may be involved in the catalysis of ADP-ribosylation.