Chemical Modification of Papain and Subtilisin: An Active Site Comparison. An Undergraduate Biochemistry Experiment

Abstract

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 proteas...