Active-site mutations of diphtheria toxin. Tryptophan 50 is a major determinant of NAD affinity.
The two active-site tryptophans of diphtheria toxin, Trp-50 and Trp-153, were individually or jointly replaced with phenylalanine or alanine by directed mutagenesis of a synthetic gene for the toxin's catalytic A fragment. Substitution of Trp-50 with alanine (W50A) decreased the ADP-ribosyltransferase activity by nearly 10(5)-fold and reduced NAD-glycohydrolase activity beyond the limits of our detection. Effects of the W153A mutation on these activities were less dramatic, < 40-fold decrease in ADP-ribosylation and < 10-fold decrease in NAD glycohydrolysis. The W50F and W153F substitutions caused only minimal reductions (< 2-fold) in enzyme activities and NAD affinity. Decreases in affinity for NAD in the initial, ground state complex, as measured by intrinsic protein fluorescence, correlated well with the reductions in enzyme activity. None of the mutations caused greater than a 10-fold decrease in NAD affinity for the ternary Michaelis complex in the ADP-ribosylation reaction; and none caused significant increase in susceptibility to proteolytic digestion by trypsin. The results indicate that Trp-50 is a major determinant of NAD affinity. Also, they identify this residue as a candidate for modification in the development of inactive forms of the toxin for use in vaccine development.
Wilson
B A
BA
Department of Microbiology and Molecular Genetics, Shipley Institute of Medicine, Boston, Massachusetts 02115.
Blanke
S R
SR
Reich
K A
KA
Collier
R J
RJ
D001665:Binding Sites / Q000235:genetics; D004167:Diphtheria Toxin / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
D004167:Diphtheria Toxin; D009243:NAD; D014364:Tryptophan; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase