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Published on: September 4, 2011
Mutagenesis and kinetic analysis of the active site Glu177 of ricin A-chain
1Department of Biological Sciences, University of Warwick, Coventry, UK.
Abstract:
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 Km 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.
Insights
Mutations in ricin A-chain (RTA) reveal the critical role of Glu177. Substituting Glu177 with Asp significantly impairs RTA
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ricin A-chain (RTA) is a potent N-glycosidase that inhibits protein synthesis by inactivating eukaryotic ribosomes.
- The enzyme removes a specific adenine residue from large rRNA, leading to cell death.
Purpose of the Study:
- To investigate the impact of mutations at the conserved active site residue Glu177 on ricin A-chain (RTA) enzyme activity.
- To elucidate the role of Glu177 in the catalytic mechanism and substrate binding of RTA.
Main Methods:
- Site-directed mutagenesis was used to create Glu177 mutants (Lys, Ala, Asp).
- Mutant protein activity was screened using an in vitro translation system.
- The Glu177Asp mutant was purified from E. coli and its kinetic properties (Km, kcat) were analyzed using yeast ribosomes.
Main Results:
- Mutagenesis of Glu177 to Lys resulted in apparent complete inactivation.
- Glu177 to Ala mutation had a minor effect, while Glu177 to Asp caused a significant reduction in activity.
- Kinetic analysis of Glu177Asp showed unchanged Km but a 49-fold decrease in kcat compared to wild-type RTA.
Conclusions:
- Glu177 is crucial for the catalytic efficiency of ricin A-chain (RTA), predominantly influencing the rate-limiting step of the enzymatic reaction.
- The results suggest Glu177 is not primarily involved in substrate binding but rather in the catalytic turnover.
- These findings provide insights into the RTA enzymatic mechanism and the functional significance of active site residues.
