Biomaterial Database

Designing substrate specificity by protein engineering of electrostatic interactions. 1987

J A Wells, and D B Powers, and R R Bott, and T P Graycar, and D A Estell

Protein engineering of electrostatic interactions between charged substrates and complementary charged amino acids, at two different sites in the substrate binding cleft of the protease subtilisin BPN', increases kcat/Km toward complementary charged substrates (up to 1900 times) and decreases kcat/Km toward similarly charged substrates. From kinetic analysis of 16 mutants of subtilisin and the wild type, the average free energies for enzyme-substrate ion-pair interactions at the two different sites are calculated to be -1.8 +/- 0.5 and -2.3 +/- 0.6 kcal/mol (1 cal = 4.18 J) [at 25 degrees C in 0.1 M Tris X HCl (pH 8.6)]. The combined electrostatic effects are roughly additive. These studies demonstrate the feasibility for rational design of charged ligand binding sites in proteins by tailoring of electrostatic interactions.

UI	MeSH Term	Description	Entries
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
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
D004563	Electrochemistry	The study of chemical changes resulting from electrical action and electrical activity resulting from chemical changes.	Electrochemistries
D005796	Genes	A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms.	Cistron,Gene,Genetic Materials,Cistrons,Genetic Material,Material, Genetic,Materials, Genetic
D005818	Genetic Engineering	Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.	Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
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