Glucosamine-6-phosphate synthase from Escherichia coli: determination of the mechanism of inactivation by N3-fumaroyl-L-2,3-diaminopropionic derivatives.
A mechanistic investigation of the inactivation of Escherichia coli glucosamine-6-phosphate synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionate (FMDP) was undertaken. On the basis of the known participation of the N-terminal cysteine residue in this process [Chmara et al. (1986) Biochim. Biophys. Acta 870, 357; Badet et al. (1988) Biochemistry 27, 2282], the model reactions between FMDP and L-cysteine and between FMDP and the synthetic decapeptide Cys-Gly-Ile-Val-Gly-Ala-Ile-Ala-Gln-Arg, corresponding to the amino-terminal protein sequence, were studied. The results allowed us to propose a pathway that is in perfect agreement with the biochemical results: enzyme inactivation arose from Michael addition of glutamine binding site cysteine-1 on the fumaroyl double bond at the beta-position of the ester group. Upon denaturation under slightly alkaline conditions, this adduct underwent cyclization to a transient succinimide adduct, which rearranged into the stable 2-substituted 1,4-thiazin-3-one-5-carboxylate involving participation of the cysteine amino group. The tryptic radiolabeled peptides purified from [3H]FMDP-treated enzyme and resistant to Edman degradation coeluted with the products resulting from the model reaction between the synthetic decapeptide and the inhibitor.
Kucharczyk
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N
Laboratoire de Bioorganique et Biotechnologies, UA CNRS 1389, ENSCP, Paris, France.
Denisot
M A
MA
Le Goffic
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F
Badet
B
B
D000409:Alanine / Q000031:analogs & derivatives*; D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005650:Fumarates / Q000032:analysis / Q000494:pharmacology*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000032:analysis / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D000637:Transaminases / Q000037:antagonists & inhibitors*; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology
D005650:Fumarates; D015091:beta-Alanine; C011757:2,3-diaminopropionic acid; C056276:N(3)-(4-methoxyfumaroyl)-2,3-diaminopropionic acid; D000637:Transaminases; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D000409:Alanine