Chemical Modification of Papain and Subtilisin: An Active Site Comparison. An Undergraduate Biochemistry Experiment
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Mireille St-Vincent [1] ; Michael Dickman [1]
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[1] Collège Universitaire de Saint-Boniface, Canada
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- Localización: Journal of chemical education, ISSN 0021-9584, Vol. 81, Nº 7, 2004, pág. 1048
- Idioma: inglés
- Texto completo no disponible (Saber más ...)
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Resumen
This experiment demonstrates the specific chemistry of cysteine and serine residues in the active sites of papain and subtilisin. While both protease enzymes catalyze the same reaction on similar substrates, papain uses cysteine to cleave peptide bonds and subtilisin employs serine for the same transformation. Treatment of both enzymes with methyl methanethiosulfonate and phenylmethylsulfonyl fluoride modifies papain and subtilisin, respectively, rendering them inactive. Methyl methanethiosulfonate reacts rapidly and exclusively with available thiols to form mixed disulfides. In the case of papain, the sole available thiol is cysteine-25 in the active site. Once this residue is modified, no further enzymatic activity is observed. Interestingly, the free thiol at cysteine-25 can be easily regenerated from the mixed disulfide, and the return of efficient catalysis can be observed. Phenylmethylsulfonyl fluoride reacts irreversibly with activated serines resulting in near complete inhibition of serine proteases. Subtilisin is modified at serine-221 to give the sulfonic ester rendering the enzyme ineffective at forming an acyl–enzyme complex with a substrate. The comparison of papain and subtilisin highlights the active site differences between the two proteases. The entire experiment can be completed in two, three-hour laboratory periods, or it could be shortened to a single, three-hour period.
