Biomaterial Database

Characterization of Escherichia coli thioredoxins with altered active site residues. 1990

F K Gleason, and C J Lim, and M Gerami-Nejad, and J A Fuchs

Department of Plant Biology, University of Minnesota, St. Paul 55108.

Escherichia coli thioredoxin is a small disulfide-containing redox protein with the active site sequence Cys-Gly-Pro-Cys-Lys. Mutations were made in this region of the thioredoxin gene and the mutant proteins expressed in E. coli strains lacking thioredoxin. Mutant proteins with a 17-membered or 11-membered disulfide ring were inactive in vivo. However, purified thioredoxin with the active site sequence Cys-Gly-Arg-Pro-Cys-Lys is still able to serve as a substrate for thioredoxin reductase and a reducing agent in the ribonucleotide reductase reaction, although with greatly reduced catalytic efficiency. A smaller disulfide ring, with the active site sequence Cys-Ala-Cys, does not turn over at a sufficient rate to be an effective reducing agent. Strain in the small ring favors the formation of intermolecular disulfide bonds. Alteration of the invariant proline to a serine has little effect on redox activity. The function of this residue may be in maintaining the stability of the active site region rather than participation in redox activity or protein-protein interactions. Mutation of the positively charged lysine in the active site to a glutamate residue raises the Km values with interacting enzymes. Although it has been proposed that the positive residue at position 36 is conserved to maintain the thiolate anion on Cys-32 (Kallis & Holmgren, 1985), the presence of the negative charge at this position does not alter the pH dependence of activity or fluorescence behavior. The lysine is most likely conserved to facilitate thioredoxin-protein interactions.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008954	Models, Biological	Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment.	Biological Model,Biological Models,Model, Biological,Models, Biologic,Biologic Model,Biologic Models,Model, Biologic
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
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
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
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
D001426	Bacterial Proteins	Proteins found in any species of bacterium.	Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
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
D013050	Spectrometry, Fluorescence	Measurement of the intensity and quality of fluorescence.	Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence