Biomaterial Database

Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase. 1995

O Cochet, and M A Badet-Denisot, and J L Teillaud, and B Badet

Laboratoire de Biotechnologie des Anticorps, Institut Curie, Paris, France.

In the present work, we attempt to identify inhibitory monoclonal antibodies directed against Escherichia coli glucosamine-6P synthase (GlmS) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis. Four of the 15 monoclonal antibodies have been shown to be specific for the native form of the enzyme and 2 of them, 505.1 and 522.2, strongly inhibit the glucosamine synthase activity. Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a Ki = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inhibitor reported so far. The reactivity of all the monoclonal antibodies with 601 overlapping octapeptides covering the entire sequence of GlmS was tested by enzyme-linked immunosorbent assay for precise epitope mapping. Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this approach. Neither 505.1 nor 522.2 was directed against linear epitopes. However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by competition experiments with an irreversible inhibitor. The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conserved among the six glucosamine-6P synthases reported so far, allowed the detection of the human enzyme.

UI	MeSH Term	Description	Entries
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
D009842	Oligopeptides	Peptides composed of between two and twelve amino acids.	Oligopeptide
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D011489	Protein Denaturation	Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein.	Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D003429	Cross Reactions	Serological reactions in which an antiserum against one antigen reacts with a non-identical but closely related antigen.	Cross Reaction,Reaction, Cross,Reactions, Cross
D004791	Enzyme Inhibitors	Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.	Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D004797	Enzyme-Linked Immunosorbent Assay	An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed.	ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
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
D005945	Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)	An enzyme that catalyzes the synthesis of fructose-6-phosphate plus GLUTAMINE from GLUTAMATE plus glucosamine-6-phosphate.	Glucosamine Synthetase,Glucosaminephosphate Isomerase (Glutamine-Forming),Hexosephosphate Aminotransferase,2-Amino-2-Deoxy-D-Glucose-6-Phosphate Ketol-Isomerase,Glucosamine 6-Phosphate Synthetase,Glucosamine-6-Phosphate Synthase,Glucosaminephosphate Isomerase (Glutamine Forming),Glutamine-Fructose-6-P Aminotransferase,Glutamine-Fructose-6-Phosphate Aminotransferase,Glutamine:Fructose-6-Phosphate-Amidotransferase,2 Amino 2 Deoxy D Glucose 6 Phosphate Ketol Isomerase,6-Phosphate Synthetase, Glucosamine,Aminotransferase, Glutamine-Fructose-6-P,Aminotransferase, Glutamine-Fructose-6-Phosphate,Aminotransferase, Hexosephosphate,Glucosamine 6 Phosphate Synthase,Glucosamine 6 Phosphate Synthetase,Glutamine Fructose 6 P Aminotransferase,Glutamine Fructose 6 Phosphate Aminotransferase,Glutamine:Fructose 6 Phosphate Amidotransferase,Ketol-Isomerase, 2-Amino-2-Deoxy-D-Glucose-6-Phosphate