Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered primary specificities.
In the past, several point mutations have been introduced individually into the substrate-binding site of alpha-lytic protease (EC 3.4.21.12) and shown to affect its specificity in a predictable manner [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. One of the resulting mutant enzymes (Met190Ala in the numbering system of Fujinaga et al.) [Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., & James, M. N. G. (1985) J. Mol. Biol. 183, 479-502] cleaves at large hydrophobic residues. We chose this enzyme as the parent for a library of mutant proteases. The library was constructed by effecting combinatorial random substitution of up to four other residues (Gly191, Arg192, Met213, and Val218) thought likely to influence the primary specificity of the protease. Active enzymes in the library were screened with a range of synthetic substrates (encompassing 19 different amino acids in the P1 position) in order to evaluate their primary cleavage preferences. The amino acid sequences of active mutants revealed a strong preference for the replacement of Met213 with a His residue. This substitution also had the greatest observed effect on specificity, conferring a greatly increased and, in some cases, dominant ability to cleave at His residues in synthetic amide substrates. Mutant enzymes with greatly increased proteolytic activity were also found in the library.
C.S.I.R.O. Division of Biomolecular Engineering, North Ryde, New South Wales, Australia.
L D
LD
Graham
K D
KD
Haggett
P A
PA
Jennings
D S
DS
Le Brocque
R G
RG
Whittaker
P A
PA
Schober
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase