Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E.
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 K(m) values of these two mutants were similar to those of the wild-type enzyme, but their k(cat) values were greatly decreased. The K(i) 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.
Liu
Jian
J
Departments of Pharmacology and Oral Surgery I, Graduate School of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
Tsukuba
Takayuki
T
Okamoto
Kuniaki
K
Ohishi
Masamichi
M
Yamamoto
Kenji
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020059:Cathepsin E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D020059:Cathepsin E