Glucosamine synthetase from Escherichia coli: kinetic mechanism and inhibition by N/sup 3/-fumaroyl-L-2,3-diaminopropionic derivatives
N/sup 3/-(4-Methoxyfumaroyl)-L-2,3-diaminopropionic acid (FMDP; 1, R = OMe), a member of a new class of glutamine analogues, has been investigated as an inhibitor of pure Escherichia coli glucosamine synthetase. Product and dead-end inhibition studies indicate an ordered association to the enzyme with the sugar molecule binding prior to substrate or inhibitor. The inactivation exhibits pseudo-first-order kinetics, is irreversible, and occurs faster in the presence of fructose 6-phosphate, a behavior previously reported for the partially purified enzyme from Salmonella typhimurium. Inhibition occurs with partial covalent incorporation of L-FMDP into glucosamine synthetase. In the presence of fructose 6-phosphate, enzyme inactivation with (2-/sup 3/H)-DL-FMDP is associated with the incorporation of 0.75 equiv of inhibitor and with the modification of 0.78 thiol residue per enzyme subunit. This result is the first evidence for covalent entrapment of the entire inhibitor molecule following FMDP-mediated glucosamine synthetase inactivation. Preliminary inactivation with 6-diazo-5-oxo-L-norleucine, known to alkylate selectively the NH/sub 2/-terminal cysteine residue, completely prevents radioactivity incorporation. Therefore, this inhibitor is postulated to covalently modify glucosamine synthetase through direct addition of the thiol nucleophile from the terminal cysteine residue to the Michael acceptor 1, so acting as an affinity label rather than a mechanism-based inhibitor.
- Research Organization:
- ENSCP, Paris (France)
- OSTI ID:
- 5244164
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:7; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Glucosamine-6-phosphate synthase from Escherichia coli: Determination of the mechanism of inactivation by N sup 3 -fumaroyl-L-2,3-diaminopropionic derivatives
Interaction of the D-isomer of 4-methylene glutamate (4-MG) with an active site thiol group of. gamma. -glutamylcysteine synthetase (. gamma. -GCS)
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALKYLATION
AMINES
AMINOTRANSFERASES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CHEMICAL REACTIONS
CHROMATOGRAPHY
DERIVATIZATION
ENZYME INHIBITORS
ENZYMES
ESCHERICHIA COLI
GLUCOSAMINE
HEXOSAMINES
HEXOSES
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
MONOSACCHARIDES
NITROGEN TRANSFERASES
ORGANIC COMPOUNDS
REACTION KINETICS
SACCHARIDES
SEPARATION PROCESSES
STOICHIOMETRY
THIN-LAYER CHROMATOGRAPHY
TRANSFERASES
TRITIUM COMPOUNDS