Mechanism of inactivation of alanine racemase by beta, beta, beta-trifluoroalanine
The alanine racemases are a group of PLP-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. Inactivation of the alanine racemases from the Gram-negative organism Salmonella typhimurium and Gram-positive organism Bacillus stearothermophilus with beta, beta, beta-trifluoroalanine has been studied. The inactivation occurs with the same rate constant as that for formation of a broad 460-490-nm chromophore. Loss of two fluoride ions per mole of inactivated enzyme and retention of (1-/sup 14/C)trifluoroalanine label accompany inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1 M guanidine) leads to rapid return of the initial 420-nm chromophore, followed by a slower (t1/2 approximately 30 min-1 h) loss of the fluoride ion and /sup 14/CO/sub 2/ release. At this point, reduction by NaB/sub 3/H/sub 4/ and tryptic digestion yield a single radiolabeled peptide. Purification and sequencing of the peptide reveals that lysine-38 is covalently attached to the PLP cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the PLP aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic beta-difluoro-alpha, beta-unsaturated imine provides an alternative mode of inhibition for these enzymes.
- Research Organization:
- Harvard Medical School, Boston, MA (USA)
- OSTI ID:
- 6075768
- Journal Information:
- Biochemistry; (United States), Vol. 28:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ALANINES
BIOCHEMICAL REACTION KINETICS
ISOMERASES
INHIBITION
PEPTIDES
AMINO ACID SEQUENCE
BACILLUS
BOROHYDRIDES
CARBON 14 COMPOUNDS
LYSINE
RACEMIZATION
SALMONELLA TYPHIMURIUM
SPECTROPHOTOMETRY
AMINO ACIDS
BACTERIA
BORON COMPOUNDS
CARBOXYLIC ACIDS
ENZYMES
HYDROGEN COMPOUNDS
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
REACTION KINETICS
SALMONELLA
550201* - Biochemistry- Tracer Techniques