caa a22 4500 397558708 CHVBK 20180308164810.0 cr unu---uuuuu 161202e199611 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021515 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021515 Characterization of the S1 Subsite Specificity of Aspergillopepsin I by Site-Directed Mutagenesis [Elektronische Daten] [Takahiro Shitani, Mizue Kobayashi, Eiji Ichishima] The structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1. However, API accommodates a Lys residue at P1, which leads to activation of trypsino-gen. On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and ▵S78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates. Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1. In contrast, substrates with a hydrophobic residue at P1 were effectively hydrolyzed by each mutant enzyme. These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P1 position. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence aspartic proteinase nationallicence aspergillopepsin I nationallicence site-directed mutagenesis nationallicence substrate specificity nationallicence trypsinogen activation nationallicence Shitani Takahiro Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 aut Kobayashi Mizue Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 aut Ichishima Eiji Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 aut The Journal of Biochemistry Oxford University Press 120/5(1996-11), 974-981 0021-924X 120:5<974 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021515 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021515 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Shitani Takahiro Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 aut NATIONALLICENCE 700 1- Kobayashi Mizue Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 aut NATIONALLICENCE 700 1- Ichishima Eiji Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/5(1996-11), 974-981 0021-924X 120:5<974 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford