Rat cathepsin H-catalyzed transacylation: comparisons of the mechanism and the specificity with papain-superfamily proteases.
We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.
Koga
H
H
Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka.
Mori
N
N
Yamada
H
H
Nishimura
Y
Y
Tokuda
K
K
Kato
K
K
Imoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D056655:Cathepsin H; D002403:Cathepsins / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010206:Papain / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D009842:Oligopeptides; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056655:Cathepsin H; C534128:Ctsh protein, rat; D010206:Papain