A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses.
1. Three synthetic substrates of cathepsin B (EC 3.4.22.1) with various amino acid residues at the P2 position (Cbz-Phe-Arg-NH-Mec, Cbz-Arg-Arg-NH-Mec and Cbz-Cit-Arg-NH-Mec, where Cbz represents benzyloxycarbonyl and NH-Mec represents 4-methylcoumarin-7-ylamide) were used to investigate the pH-dependency of cathepsin B-catalysed hydrolyses and to obtain information on the nature of enzyme-substrate interactions. 2. Non-linear-regression analysis of pH-activity profiles for these substrates indicates that at least four ionizable groups on cathepsin B with pKa values of 3.3, 4.55, 5.46 and greater than 7.3 can affect the rate of substrate hydrolysis. 3. Ionization of the residue with a pKa of 5.46 has a strong effect on activity towards the substrate with an arginine in P2 (8.4-fold increase in activity) but has only a moderate effect on the rate of hydrolysis with Cbz-Cit-Arg-NH-Mec (2.3-fold increase in activity) and virtually no effect with Cbz-Phe-Arg-NH-Mec. The kinetic data are consistent with this group being an acid residue with a side chain able to interact with the side chains of an arginine or a citrulline in the P2 position of a substrate. Amino acid sequence alignment and model building with the related enzyme papain (EC 3.4.22.2) suggest that Glu-245 of cathepsin B is a likely candidate. The relative importance of electrostatic and hydrophobic interactions in the S2 subsite of cathepsin B is discussed. 4. For all three substrates, activity appears after ionization of a group with a pKa of 3.3, believed to be the active-site Cys-29 of cathepsin B. The identity of the groups with pKa values of 4.55 and greater than 7.3 remains unknown.
Khouri
H E
HE
Protein Engineering Section, National Research Council Canada, Montréal, Québec.
Plouffe
C
C
Hasnain
S
S
Hirama
T
T
Storer
A C
AC
Ménard
R
R
D002401:Cathepsin B / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013379:Substrate Specificity
D003374:Coumarins; D004151:Dipeptides; D005456:Fluorescent Dyes; C069014:benzyloxycarbonylcitrullyl-arginine 4-methylcoumarin-7-ylamide; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D002401:Cathepsin B