Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding |
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Authors: | Andy Chevigné Marie-Eve Dumez Mireille Dumoulin André Matagne Alain Jacquet Moreno Galleni |
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Institution: | 1. Macromolécules Biologiques, Centre d''Ingénierie des Protéines, Université de Liège, Institut de Chimie B6, Sart Tilman Liège B-4000, Belgique;2. Laboratoire d''Allergologie Experimentale, Université Libre de Bruxelles, IBMM, Gosselies B-6041, Belgique;3. Laboratoire d''Enzymologie, Centre d''Ingénierie des Protéines, Université de Liège, Institut de Chimie B6, Sart Tilman Liège B-4000, Belgique |
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Abstract: | BackgroundPapain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes.MethodsTo have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1.ResultsWe showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen.General significanceFinally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis. |
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Keywords: | AMC 7-amino-4-methylcoumarin ANS 8-anilino-1-naphthalenesulfonic acid BMGY Yeast Glycerol Buffered Media Boc N-t-butoxy-carbonyl C34A replacement of the catalytic cysteine (C34) by an alanine DTT dithiotreitol E-64 L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane EDTA ethylene diamine tetra acetic PBS phosphate buffered saline PDC pentadentate chelator p propeptide numbering proDer p 1 Der p 1 zymogen prop propeptide SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis WT wild-type |
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