Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide).
Cathepsin B (EC 3.4.22.1) from bovine spleen and the analogous enzyme from rat liver were investigated at 25 degrees C at I0.1 in acidic media by kinetic study of (a) the reactions of their catalytic-site thiol groups towards the two-protonic-state reactivity probe 2,2'-dipyridyl disulphide and (b) their catalysis of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide. Reactivity-probe kinetics showed that nucleophilic character is generated in the sulphur atom of cathepsin B by protonic dissociation with pKa 3.4, presumably to form an S-/ImH+ ion-pair. Substrate-catalysis kinetics showed that ion-pair formation is not sufficient to generate catalytic competence in cathepsin B, because catalytic activity is not generated as the pH is raised across pKa 3.4 but rather as it is raised across pKa 5-6 (5.1 for kcat; 5.6 for kcat./Km for the bovine spleen enzyme and 5.8 for kcat./Km for the rat liver enzyme). The implications of these results and of known structural differences between the catalytic sites of the rat liver enzyme and papain (EC 3.4.22.2) for the mechanism of cysteine-proteinase-catalysed hydrolysis are discussed.
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives*; D001587:Benzoylarginine-2-Naphthylamide / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D002401:Cathepsin B; D002403:Cathepsins / Q000378:metabolism*; D002417:Cattle; D004220:Disulfides; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D011725:Pyridines / Q000378:metabolism*; D051381:Rats; D013045:Species Specificity; D013154:Spleen / Q000201:enzymology
D004220:Disulfides; D007202:Indicators and Reagents; D011725:Pyridines; C001105:2,2'-dipyridyl disulfide; D001587:Benzoylarginine-2-Naphthylamide; D015082:2,2'-Dipyridyl; C028469:N-benzyloxycarbonylarginyl-L-arginine-2-naphthylamide; D001120:Arginine; D002403:Cathepsins; D002401:Cathepsin B