Biomaterial Database

Variants of subtilisin BPN' with altered specificity profiles. 1994

M Rheinnecker, and J Eder, and P S Pandey, and A R Fersht

MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.

A strategy for increasing the size of the S4 binding pocket was used to improve the specificity of subtilisin BPN' toward substrates with large hydrophobic P4 side chains. This approach involves single and double amino acid replacements at positions 104, 107, and 126. Previously, alteration of I107 to glycine has been found to increase the specificity of subtilisin toward leucine, isoleucine, and phenylalanine as P4 residues by up to 214-fold. Replacement of Y104 by alanine also yields a similar improvement in specificity. However, this subtilisin variant favors isoleucine and phenylalanine over leucine. When L126 was replaced by valine, alanine, and glycine, respectively, only the L126A subtilisin variant, which possesses a 28-fold-increased catalytic efficiency for isoleucine compared with all other substrates tested, showed a significantly improved specificity profile. As inferred from the double-mutant enzymes I107G/L126V, I107G/L126A, and I107G/Y104A, none of the effects of the single amino acid replacements on the kinetic parameters are additive. The I107G/L126V mutant subtilisin has the largest improvement in P4 substrate specificity reported so far: kcat/KM is increased 340-fold for leucine compared to alanine. By contrast, the specificity profile of the I107G/Y104A mutant enzyme is impaired in comparison with that of the corresponding single mutants. Therefore, the design of high-specificity subtilisin variants through the combination of single amino acid replacements in the S4 pocket appears to be nontrivial due to the interference of the introduced structural changes.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D013379	Substrate Specificity	A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.	Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D013381	Subtilisins	A family of SERINE ENDOPEPTIDASES isolated from Bacillus subtilis. EC 3.4.21.-	Alcalase,AprA-Subtilisin,Bacillus amyloliquefaciens Serine Protease,Bacillus subtilis Alkaline Proteinase,Carlsberg Subtilisin,Maxatase,Nagarse,Novo Alcalase,Profezim,Protease VII,Subtilisin 72,Subtilisin A,Subtilisin BPN',Subtilisin Carlsberg,Subtilisin DY,Subtilisin E,Subtilisin GX,Subtilisin Novo,Subtilopeptidase A,Alcalase, Novo,AprA Subtilisin,Subtilisin, Carlsberg
D014644	Genetic Variation	Genotypic differences observed among individuals in a population.	Genetic Diversity,Variation, Genetic,Diversity, Genetic,Diversities, Genetic,Genetic Diversities,Genetic Variations,Variations, Genetic
D016297	Mutagenesis, Site-Directed	Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.	Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses