Site-directed mutagenic alteration of potential active-site residues of the A subunit of Escherichia coli heat-labile enterotoxin. Evidence for a catalytic role for glutamic acid 112.

Escherichia coli heat-labile enterotoxin (LT) and the related cholera toxin exert their effects on eukaryotic cells through the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex. The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicinal to a presumptive NAD(+)-binding site and thus may play a role in substrate binding or catalysis. Using a plasmid clone encoding the A subunit of LT, we have introduced substitutions at such potential active-site residues and analyzed the enzymatic properties of the resultant mutant analogs. Enzymatic analyses, employing both transducin and agmatine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzyme activity to < 10% of wild-type levels. Kinetic analyses indicated that alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD+ or agmatine. Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative effects on kcat and kcat/Km. The results provide formal evidence that glutamic acid 112 of the A subunit of LT represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD+ susceptible to nucleophilic attack by an incoming acceptor substrate.