The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family. In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+. Glu-129 was previously shown to be critical for both enzymatic activities. In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129. The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses. Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1. This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way. In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1. These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates.