Biomaterial Database

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8-azido-AMP. 1988

R V Patil, and P Datta

Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.

The photoreactive AMP analog, 8-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 microM and 1.5 microM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Photolabeling of AMP-free dehydratase with 8-azido-[2-3H]AMP resulted in a time and concentration-dependent enzyme inactivation and concomitant incorporation of 8-azido-AMP into protein. At low 8-azido-AMP concentrations, incorporation of about 1 mol 8-azido-AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8-azido-AMP binding. Ultraviolet irradiation with 20 microM 3H-labeled 8-azido-AMP revealed one tryptic peptide, Thr230-Thr-Gly-Thr-Leu-Ala-Asp-Gly-Cys-Asp-Val-Ser-Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8-azido-AMP, most likely represents the AMP-binding region on the dehydratase molecule.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D002850	Chromatography, Gel	Chromatography on non-ionic gels without regard to the mechanism of solute discrimination.	Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
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
D000249	Adenosine Monophosphate	Adenine nucleotide containing one phosphate group esterified to the sugar moiety in the 2'-, 3'-, or 5'-position.	AMP,Adenylic Acid,2'-AMP,2'-Adenosine Monophosphate,2'-Adenylic Acid,5'-Adenylic Acid,Adenosine 2'-Phosphate,Adenosine 3'-Phosphate,Adenosine 5'-Phosphate,Adenosine Phosphate Dipotassium,Adenosine Phosphate Disodium,Phosphaden,2' Adenosine Monophosphate,2' Adenylic Acid,5' Adenylic Acid,5'-Phosphate, Adenosine,Acid, 2'-Adenylic,Acid, 5'-Adenylic,Adenosine 2' Phosphate,Adenosine 3' Phosphate,Adenosine 5' Phosphate,Dipotassium, Adenosine Phosphate,Disodium, Adenosine Phosphate,Monophosphate, 2'-Adenosine,Phosphate Dipotassium, Adenosine,Phosphate Disodium, Adenosine
D000345	Affinity Labels	Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids.	Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D000495	Allosteric Site	A site on an enzyme which upon binding of a modulator, causes the enzyme to undergo a conformational change that may alter its catalytic or binding properties.	Allosteric Sites,Site, Allosteric,Sites, Allosteric
D001386	Azides	Organic or inorganic compounds that contain the -N3 group.	Azide
D013913	Threonine Dehydratase	A pyridoxal-phosphate protein that catalyzes the deamination of THREONINE to 2-ketobutyrate and AMMONIA. The role of this enzyme can be biosynthetic or biodegradative. In the former role it supplies 2-ketobutyrate required for ISOLEUCINE biosynthesis, while in the latter it is only involved in the breakdown of threonine to supply energy. This enzyme was formerly listed as EC 4.2.1.16.	Threonine Deaminase,Threonine Dehydrase,Threonine Ammonia-Lyase,Ammonia-Lyase, Threonine,Deaminase, Threonine,Dehydrase, Threonine,Dehydratase, Threonine,Threonine Ammonia Lyase
D014357	Trypsin	A serine endopeptidase that is formed from TRYPSINOGEN in the pancreas. It is converted into its active form by ENTEROPEPTIDASE in the small intestine. It catalyzes hydrolysis of the carboxyl group of either arginine or lysine. EC 3.4.21.4.	Tripcellim,Trypure,beta-Trypsin,beta Trypsin