| Active-site mutations of diphtheria toxin. Tryptophan 50 is a major determinant of NAD affinity. | |
| | |
MedLine Citation:
|
PMID: 8083236 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
|
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. |
| | |
Authors:
|
B A Wilson; S R Blanke; K A Reich; R J Collier |
Related Documents
:
|
163226 - Erythritol catabolism by brucella abortus. 12083366 - Nad-dependent inhibition of the nad-glycohydrolase activity in a549 cells. 2764986 - Interconversion of nad(h) to nadp(h). a cellular response to quinone-induced oxidative ... 7263646 - Inhibition of the mitochondrial nicotinamide nucleotide transhydrogenase by dicyclohexy... 20013386 - The use of liposomes in the study of drug metabolism: a method to incorporate the enzym... 10664846 - A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, armillar... |
Publication Detail:
|
Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
|
Title: The Journal of biological chemistry Volume: 269 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1994 Sep |
Date Detail:
|
Created Date: 1994-10-11 Completed Date: 1994-10-11 Revised Date: 2007-11-14 |
Medline Journal Info:
|
Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
|
Languages: eng Pagination: 23296-301 Citation Subset: IM |
Affiliation:
|
Department of Microbiology and Molecular Genetics, Shipley Institute of Medicine, Boston, Massachusetts 02115. |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
Binding Sites
/
genetics Diphtheria Toxin / genetics*, metabolism Kinetics Mutagenesis, Site-Directed* NAD / metabolism* NAD+ Nucleosidase / metabolism Poly(ADP-ribose) Polymerases / metabolism Spectrometry, Fluorescence Tryptophan / metabolism* |
| Grant Support | |
ID/Acronym/Agency:
|
AI08315/AI/NIAID NIH HHS; AI22021/AI/NIAID NIH HHS; AI22848/AI/NIAID NIH HHS |
| Chemical | |
Reg. No./Substance:
|
0/Diphtheria Toxin; 53-84-9/NAD; 73-22-3/Tryptophan; EC 2.4.2.30/Poly(ADP-ribose) Polymerases; EC 3.2.2.5/NAD+ Nucleosidase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of inte...
Next Document: Heterogeneity of ternary complex factors in HeLa cell nuclear extracts.