Multiple proteolytic action of rat liver cathepsin B: specificities and pH-dependences of the endo- and exopeptidase activities.
Dipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The eipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the alpha-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated alpha-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the alpha-imino moiety and the protonated or amidated alpha-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.
Koga
H
H
Faculty of Pharmaceutical Science, Kyushu University, Fukuoka.
Yamada
H
H
Nishimura
Y
Y
Kato
K
K
Imoto
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism; D020689:Exopeptidases; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000596:Amino Acids; D010450:Endopeptidases; D020689:Exopeptidases; D010447:Peptide Hydrolases; D002401:Cathepsin B