title: Mechanism of the reaction of papain with substrate-derived diazomethyl ketones. Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism.
abstract: The reactions of papain (EC 3.4.22.2) with substrate-derived diazomethyl ketones reported by Leary, Larsen, Watanabe & Shaw [Biochemistry (1977) 16, 5857--5861] are unusual in that (i) these reagents fail to react with low-molecular-weight thiols and (ii) the rate of reaction with the papain thiol group does not decrease to near-zero values across a pKa of 4 as the pH is decreased. Existing data are shown to suggest an interpretation involving neighbouring-group participation via transient thiohemiketal formation, rate-determining protonation by imidazolium ion and alkylation on sulphur via a three-membered cyclic transition state. Implications for (a) the difference in site-specificity exhibited by halomethyl ketones in their reactions with serine proteinases and cysteine proteinases and (b) stereoelectronic requirements in the mechanism of papain-catalysed hydrolysis are discussed. The possibility of two tetrahedral intermediates between adsorptive complex and acyl-enzyme is indicated.
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D007659:Ketones / Q000378:metabolism; D008956:Models, Chemical; D008968:Molecular Conformation; D010206:Papain / Q000378:metabolism*; D012694:Serine
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D007659:Ketones; C015795:diazomethyl ketones; D012694:Serine; D003978:Diazomethane; D010450:Endopeptidases; D010206:Papain; D003545:Cysteine