Identification of the Escherichia coli ADP-glucose synthetase inhibitor binding site(s)
The photoaffinity labeling agent 8-azido adenylate (AMP) is an inhibitor site specific probe of the E. coli ADPG synthetase. In the absence of light, 8-azido AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), (2-/sup 3/H)8-azido AMP specifically and covalently incorporates into the enzyme. Photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADP-glucose (ADPG) provides nearly 100% protection from 8-azido AMP photoinactivation, while the substrate AMP provides approximately 50% protection and the inhibitor AMP provides approximately 30% protection. These three adenylate allosteric effects of E. coli ADPG synthetase also protect it from photoincorporation of 8-azido AMP. The reaction site(s) of (2-/sup 3/H)-azido AMP with the enzyme was identified by reverse phase HPLC isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease generated peptides containing the labeled analog. This site is the same as the major binding region of the substrate site specific probe, 8-azido ADP-(/sup 14/C)glucose. Conformational analysis of this region predicts that it is a part of a Rossmann fold, the super-secondary structure found in many adenine nucleotide binding proteins. Two minor reaction regions of the enzyme with (2-/sup 3/H)8-azido AMP were also identified. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure.
- Research Organization:
- California Univ., Davis (USA)
- OSTI ID:
- 5593608
- Country of Publication:
- United States
- Language:
- English
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Cells
& Tissue Culture
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ALDEHYDES
AMP
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CARBOHYDRATES
CARBON 14 COMPOUNDS
CONFIGURATION INTERACTION
ELECTROMAGNETIC RADIATION
ENZYME INHIBITORS
ENZYMES
ESCHERICHIA COLI
EXTREME ULTRAVIOLET RADIATION
GLUCOSE
HEXOSES
KINETICS
LABELLED COMPOUNDS
LIGASES
MICROORGANISMS
MONOSACCHARIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
RADIATION EFFECTS
RADIATIONS
REACTION KINETICS
SACCHARIDES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION