Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues.
The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with Glu and Asp to introduce a high specificity for basic residues into the S1' site of trypsin. Both mutations cause a dramatic increase in the S1' specificity for Arg and Lys as measured by acyl transfer reactions. In K60E, the preference for Arg increases 70-fold while the preference for P1'-Lys increases 12-fold. In contrast, the preferences for other P1' residues either decrease slightly or remain the same. Thus, K60E prefers P1'-Arg over most other P1' residues by 2 orders of magnitude. Similar results are obtained when P1' specificity is measured in peptide cleavage assays. K60D exhibits an S1' specificity profile very similar to that of K60E, although the P1'-Arg preference is reduced by a factor of 2.5. Molecular modeling studies suggest that the high S1' specificity for Arg in K60E may be due to the formation of a salt bridge between Glu60 and the P1'-Arg of the substrate.
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
T
T
Kurth
S
S
Grahn
M
M
Thormann
D
D
Ullmann
H J
HJ
Hofmann
H D
HD
Jakubke
L
L
Hedstrom
D000599:Amino Acids, Diamino / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D014357:Trypsin / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism*
D000599:Amino Acids, Diamino; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D014357:Trypsin; D008239:Lysine