Lecture 10,16,17 - Protein engineering of subtilisin...

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Protein engineering of subtilisin Subtilisin: serine endopeptidase, MW 27,500 (275 residues). Secreted in large amounts from many Bacillus species. X-ray structures have been determined (globular protein, several ! -helices).
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Protein engineering of subtilisin Genetically unrelated to mammalian serine proteases, but has a similar active site structure. Active site features a charge- relay network involving Asp-32, His-64, and active site Ser-221.
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Charge-relay site of subtilisin Asp-32 carboxylate side chain H- bonds to a N-bonded proton on His- 64 imidazole ring. The other N-bonded proton on His-64 H-bonds to the O-H proton of Ser- 221.
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Charge-relay site of subtilisin Ser-221 becomes ‘activated’ by virtue of charge-separation of O-H, and its O-atom becomes more nucleophilic. Ser-221 O-atom attacks incoming substrate, assisted by a neighboring carboxyamide side chain of Asn-155.
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Charge-relay site of subtilisin Substrate is a susceptible peptide bond bound into the active site cleft. Action: Substrate peptide bond is cleaved.
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Catalytic triad of subtilisin E - S E - S !
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Mutagenesis of subtilisin Researchers were interested in: Determining residues involved in catalysis. Enhancing enzyme stability. Obtaining details of substrate specificity. Determining effect of disulfide bonds on secretion, structure, and stability.
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Cassette mutagenesis of subtilisin Method: choose a codon in the gene and perform ‘randomized mutagenesis’. Use 25% each of nucleotides A, C, T, G at each of three codon bases during oligonucleotide synthesis. By this method, any three-base codon can arise.
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Cassette mutagenesis of subtilisin Goal: engineering of oxidative resistance. Subtilisin known to be stable to denaturants, but highly susceptible to chemical oxidation. Peptide mapping studies showed that oxidizing Met-222 (next to catalytic Ser-221) results in 90% loss of enzyme activity.
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Methionine oxidized to methionine sulfoxide: -CH 2 CH 2 S-CH 3 -CH 2 CH 2 S-CH 3 O
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Cassette mutagenesis of subtilisin Met-222 is a conserved residue in all subtilisins, suggesting functional role. This is proved by the fact that the enzyme is easily inactivated by oxidizing Met-222. Can oxidative loss be prevented by replacing Met with a residue insensitive to oxygen (while retaining enzyme function)?
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Active site of subtilisin Met-222 is adjacent to active site residues
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Mutagenesis of subtilisin Protein engineering experiments: All 19 aa’s were produced at subtilisin codon 222. All were satisfactorily expressed by B. subtilis (note this protein is native to this organism). All 19 subtilisin mutants were purified, and specific activity of each was determined.
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of codon-222 mutant subtilisins Codon-222 Cys Met Ala Ser Gly Thr Asn Pro Leu Val Percent relative specific activity 138.0 100.0 53.0 35.0 30.0 28.0 15.0 13.0 12.0 9.3 Wells et al , 1985 Codon-222 Gln Phe
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Lecture 10,16,17 - Protein engineering of subtilisin...

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