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
N1 - Funding Information:
Animal members of the pepsin family include secretory enzymes such as pepsin and renin and nonsecretory enzymes such as cathepsin D and cathepsin E. They are known to be composed of two homologous domains, despite having very little amino acid sequence similarity (reviewed in Refs. 1- 3). Each domain contains one of the pair of catalytic Asp residues which reside in a consensus D(T/S)G motif Cathepsin E is a major intracellular aspartic protease that exists as a homodimer of two catalytically identical monomers with a molecular mass of 42 kDa (4-8). Among the pepsin family of aspartic proteinases, cathepsin E is unique in its limited tissue distribution in certain cell types such as lymphoid tissues, gastrointestinal tracts, urinary organs, and blood cells (9, 10) and its cell-specific localization (11-14). Cathepsin E is also unique in its structure. The amino terminal portion contains a Cys residue at position 43, which 'This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. 2To whom correspondence should be addressed. Tel: +81-92-642-6337, Fax: +81-92-642-6342, E-mail: kyama@dent.kyushu-u.acjp Abbreviations: CD, circular dichroism; DMEM, Dulbecco's modified Eagle* medium; FCS, fetal calf serum; Dnp iV-2,4^trophenyl; MOCAc, (7-methoxycoumann^-yl)acetyl; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
PY - 2002/9
Y1 - 2002/9
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
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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 -