A remodelled protease that cleaves phosphotyrosine substrates

被引:18
作者
Knigh, Zachary A.
Garrison, Jennifer L.
Chan, Karina
King, David S.
Shokat, Kevan M. [1 ]
机构
[1] Univ Calif San Francisco, Howard Hughes Med Inst, Dept Cell & Mol Pharmacol, San Francisco, CA 94107 USA
[2] Univ Calif Berkeley, Howard Hughes Med Inst, Dept Chem, Berkeley, CA 94720 USA
关键词
D O I
10.1021/ja073875n
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Proteases have evolved to recognize a diverse range of polypeptide sequences. Remarkably, no protease is known that selectively cleaves its substrates adjacent to a site of post-translational modification. To explore the requirements for such a catalyst, we sought to identify mutations that would convert the bacterial protease subtilisin BPN', which preferentially cleaves peptides at phenylalanine or tyrosine residues, into a phosphotyrosine selective protease. Through iterative modeling, mutagenesis, and kinetic analysis, we have identified a subtilisin variant (E156R/P129G) that preferentially cleaves phosphotyrosine peptides relative to the optimal wildtype substrates. This double mutant achieves a 2500-fold enhancement in phosphotyrosine selectivity relative to the wild-type enzyme through the introduction of complementary steric and electrostatic features.
引用
收藏
页码:11672 / +
页数:3
相关论文
共 50 条
[41]   Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein [J].
Lin, XL ;
Koelsch, C ;
Wu, SL ;
Downs, D ;
Dashti, A ;
Tang, J .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (04) :1456-1460
[42]   Plasmodium falciparum-derived cysteine protease cleaves human erythrocyte ankyrin. [J].
Hanspal, M ;
Liu, SC ;
Chishti, A .
BLOOD, 1998, 92 (10) :4A-4A
[43]   A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma [J].
Brossier, F ;
Jewett, TJ ;
Sibley, LD ;
Urban, S .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2005, 102 (11) :4146-4151
[44]   The Nuclear Inclusion a (NIa) Protease of Turnip Mosaic Virus (TuMV) Cleaves Amyloid-β [J].
Han, Hye-Eun ;
Sellamuthu, Saravanan ;
Shin, Bae Hyun ;
Lee, Yong Jae ;
Song, Sungmin ;
Seo, Ji-Seon ;
Baek, In-Sun ;
Bae, Jeomil ;
Kim, Hannah ;
Yoo, Yung Joon ;
Jung, Yong-Keun ;
Song, Woo Keun ;
Han, Pyung-Lim ;
Park, Woo Jin .
PLOS ONE, 2010, 5 (12)
[45]   Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy [J].
Badorff, C ;
Lee, GH ;
Lamphear, BJ ;
Martone, ME ;
Campbell, KP ;
Rhoads, RE ;
Knowlton, KU .
NATURE MEDICINE, 1999, 5 (03) :320-326
[46]   MEMBRANAL PROTEASE THAT CLEAVES THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE [J].
ALHANATY, E ;
SHALTIEL, S .
ISRAEL JOURNAL OF MEDICAL SCIENCES, 1979, 15 (09) :789-790
[47]   The serine protease trypsin cleaves C termini of β- and γ-subunits of epithelial Na+ channels [J].
Jovov, B ;
Berdiev, BK ;
Fuller, CM ;
Ji, HL ;
Benos, DJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (06) :4134-4140
[48]   Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy [J].
Cornel Badorff ;
Gil-Hwan Lee ;
Barry J. Lamphear ;
Maryann E. Martone ;
Kevin P. Campbell ;
Robert E. Rhoads ;
Kirk U. Knowlton .
Nature Medicine, 1999, 5 :320-326
[49]   Human Tdp1 cleaves a broad spectrum of substrates, including phosphoamide linkages [J].
Interthal, H ;
Chen, HJ ;
Champoux, JJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2005, 280 (43) :36518-36528
[50]   Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy [J].
Burkart, Anna D. ;
Xiong, Bo ;
Baibakov, Boris ;
Jimenez-Movilla, Maria ;
Dean, Jurrien .
JOURNAL OF CELL BIOLOGY, 2012, 197 (01) :37-44