A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and -161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B and of L-arginine 2-naphthylamide catalysed by cathepsin H.
The pH-dependences of kcat, Km and kcat./Km for the hydrolysis at 25 degrees C at I 0.1 of L-arginine 2-naphthylamide catalysed by cathepsin H from bovine spleen were determined in the pH range approx. 4-8. The pH-dependences of these kinetic parameters were determined also for the hydrolysis at 25 degrees C at I 0.1 of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B (EC 3.4.22.1) from bovine spleen in the pH range 7-8, which extends the studies in acidic media reported by Willenbrock & Brocklehurst [(1984) Biochem. J. 222, 805-814]. These results are discussed and related to those from the reactivity-probe kinetics reported in the preceding paper [Willenbrock & Brocklehurst (1985) Biochem. J. 227, 511-519] and to known structural features present in rat liver cathepsins B and H and in papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). Consideration of the kinetic data leads to the suggestion that in the cysteine proteinases rearrangement of intimate S-/ImH+ ion-pairs in catalytic sites is brought about by a combination of field effects in the immediate vicinity of the ion-pair and consequences of protonic dissociation of a group with pKa 5-6 remote from the catalytic site. The contributions of the two types of effect seem to differ from enzyme to enzyme. Of the four cysteine proteinases considered, only cathepsin B exerts an absolute requirement for the proton-deficient form of a group with pKa 5-6 for catalytic activity. Protonic dissociation with pKa 5-6 enhances catalytic activity in cathepsin H and in actinidin and appears to have little or no effect in papain. Only cathepsin B lacks a polar or negatively charged side chain in the residue analogous to Asp-158 in papain, and this is suggested to account for its total dependence on a protonic dissociation remote from the catalytic site.
D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001587:Benzoylarginine-2-Naphthylamide / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002401:Cathepsin B; D056655:Cathepsin H; D002403:Cathepsins / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010206:Papain / Q000378:metabolism*; D013053:Spectrophotometry
D001587:Benzoylarginine-2-Naphthylamide; C029772:arginine beta-naphthylamide; C028469:N-benzyloxycarbonylarginyl-L-arginine-2-naphthylamide; D001120:Arginine; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C018745:actinidain; D056655:Cathepsin H; C534128:Ctsh protein, rat; D010206:Papain