Analysis of several key active site residues of ricin A chain by mutagenesis and X-ray crystallography.
Active site residues of ricin A chain were analyzed by site-directed mutagenesis and X-ray diffraction to help assess their roles in the mechanism of action of this toxic N-glycosidase enzyme. Arg180 is thought, from X-ray studies, to protonate the adenine substrate at N3; this facilitates bond cleavage and is crucial to the mechanisms of action. The residue was converted to Gln and initial rate data measured. Km for the mutant is not significantly affected, increasing only 2-fold. The kcat, however, is decreased approximately 1000-fold. This is consistent with a simple interpretation that Arg180 is involved more in transition state stabilization than in substrate binding. Tyrosines 80 and 123 are known from X-ray models to stack on either side of the substrate adenine ring. When they were each converted to serine overall activity was reduced 160- and 70-fold respectively against ribosomes from Artemia salina. These effects are each approximately 10 times greater than when the residues were previously converted to phenylalanines. Sufficient protein for the Tyr80 to Phe mutant was obtained to carry out an X-ray analysis. Together with mutagenesis data, the structure suggests that the invariance of the two active site Tyr residues is largely caused by structural stability.
Kim
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Clayton Foundation Biochemical Institute, Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Robertus
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JD
D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects; D012276:Ricin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D012276:Ricin