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-ribosyla tion of guanine nucleotide-binding proteins of the adenylate cyclase c omplex. The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicin al to a presumptive NAD(+)-binding site and thus may play a role in su bstrate binding or catalysis. Using a plasmid clone encoding the A sub unit of LT, we have introduced substitutions at such potential active- site residues and analyzed the enzymatic properties of the resultant m utant analogs. Enzymatic analyses, employing both transducin and agmat ine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzy me activity to <10% of wild type levels, Kinetic analyses indicated th at alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD(+) or agmati ne. Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative ef fects on k(cat) and k(cat)/K-m. The results provide formal evidence th at glutamic acid 112 of the A subunit of LT represents a functional ho molog or analog of catalytic glutamic acid residues that have been ide ntified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD(+) susceptible to nucleop hilic attack by an incoming acceptor substrate.