Mutagenesis and kinetic analysis of the active site Glu177 of ricin A-chain

Ricin A-chain (RTA) is an N-glycosidase which removes a specific adenine residue from the large rRNA of eukaryotic ribosomes. As a consequence, the ribosome is inactivated and protein synthesis is inhibited leading to cell death. This report describes the effects on enzyme activity of specific mutat...

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Bibliographic Details
Published in:Protein engineering 1993-06, Vol.6 (4), p.425-431
Main Authors: Chaddock, John A., Roberts, Lynne M.
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Biological and medical sciences
Biotechnology
Cloning, Molecular
DNA - genetics
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gene Expression
Glutamine - genetics
Kinetics
Methods. Procedures. Technologies
Mutagenesis
N-glycosidase
Protein engineering
Rabbits
Reticulocytes - metabolism
Ricin - chemistry
Ricin - genetics
Ricin - metabolism
ricin A-chain
structure—function
Transcription, Genetic
Transformation, Bacterial
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Summary:Ricin A-chain (RTA) is an N-glycosidase which removes a specific adenine residue from the large rRNA of eukaryotic ribosomes. As a consequence, the ribosome is inactivated and protein synthesis is inhibited leading to cell death. This report describes the effects on enzyme activity of specific mutations of the conserved active site Glu177. The activity of mutant proteins was initially screened using an in vitro translation system. It was found that mutagenesis of Glu177 to Lys led to an apparent total inactivation of the enzyme, Glu177 to Ala had a small effect on activity, whereas the conservative Glu177 to Asp mutation had a significant effect. The properties of Glu177 to Asp were investigated more closely. Mutant protein was purified from an Escherichia coli expression system and kinetic analysis of the depurination activity assessed using salt-washed yeast ribosomes. It was shown that the K, of the mutant protein was unchanged when compared to data of wild type RTA; however, the kcat was significantly decreased (49-fold compared to wild type RTA). This suggests that Glu177 plays a predominant role in the rate-limiting step of the enzymatic mechanism and not in substrate binding. These data are discussed in relation to other reports of ricin Glu177 substitutions.
ISSN:1741-0126
0269-2139
1741-0134
1460-213X
DOI:10.1093/protein/6.4.425