Abstract
Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif. Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis. Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells. The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions. However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively. The Km values of these two mutants were similar to those of the wild-type enzyme, but their kcat values were greatly decreased. The Ki values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same. The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values. These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product.
| Original language | English |
|---|---|
| Pages (from-to) | 493-499 |
| Number of pages | 7 |
| Journal | Journal of Biochemistry |
| Volume | 132 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2002 |
| Externally published | Yes |
Fingerprint
Keywords
- Active-site motif
- Aspartic proteinase
- Cathepsin E
- Characterization of mutants
- Correct folding
ASJC Scopus subject areas
- Medicine(all)
- Biochemistry
- Molecular Biology
Cite this
Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E1. / Liu, Jian; Tsukuba, Takayuki; Okamoto, Kuniaki; Ohishi, Masamichi; Yamamoto, Kenji.
In: Journal of Biochemistry, Vol. 132, No. 3, 2002, p. 493-499.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E1
AU - Liu, Jian
AU - Tsukuba, Takayuki
AU - Okamoto, Kuniaki
AU - Ohishi, Masamichi
AU - Yamamoto, Kenji
PY - 2002
Y1 - 2002
N2 - Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif. Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis. Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells. The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions. However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively. The Km values of these two mutants were similar to those of the wild-type enzyme, but their kcat values were greatly decreased. The Ki values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same. The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values. These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product.
AB - Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif. Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis. Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells. The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions. However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively. The Km values of these two mutants were similar to those of the wild-type enzyme, but their kcat values were greatly decreased. The Ki values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same. The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values. These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product.
KW - Active-site motif
KW - Aspartic proteinase
KW - Cathepsin E
KW - Characterization of mutants
KW - Correct folding
UR - http://www.scopus.com/inward/record.url?scp=0036737865&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036737865&partnerID=8YFLogxK
U2 - 10.1093/oxfordjournals.jbchem.a003247
DO - 10.1093/oxfordjournals.jbchem.a003247
M3 - Article
C2 - 12204120
AN - SCOPUS:0036737865
VL - 132
SP - 493
EP - 499
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 3
ER -