Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution.
Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)
Abrahmsén
L
L
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
Tom
J
J
Burnier
J
J
Butcher
K A
KA
Kossiakoff
A
A
Wells
J A
JA
D000588:Amines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D015202:Protein Engineering; D013381:Subtilisins / Q000235:genetics*
D000588:Amines; D010455:Peptides; D013381:Subtilisins