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 pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)
Wilson
B A
BA
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts.
Reich
K A
KA
Weinstein
B R
BR
Collier
R J
RJ
D000246:Adenosine Diphosphate Ribose / Q000096:biosynthesis; D000345:Affinity Labels; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D004167:Diphtheria Toxin / Q000235:genetics* / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D005973:Glutamine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity
D000345:Affinity Labels; D004167:Diphtheria Toxin; D005971:Glutamates; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D005973:Glutamine; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine