Mechanism of inactivation of alanine racemase by .beta.,.beta.,.beta.-trifluoroalanine
| Autor: | Faraci Ws, Walsh Ct |
|---|---|
| Rok vydání: | 1989 |
| Předmět: |
Salmonella typhimurium
Stereochemistry Molecular Sequence Data Peptide Borohydrides Tritium Biochemistry Cofactor chemistry.chemical_compound Bacillus cereus Alanine racemase Denaturation (biochemistry) Amino Acid Sequence Carbon Radioisotopes Guanidine Racemization Amino Acid Isomerases chemistry.chemical_classification Alanine Binding Sites biology Lysine Alanine Racemase Peptide Fragments Kinetics Enzyme chemistry Spectrophotometry biology.protein |
| Zdroj: | Biochemistry. 28:431-437 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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 [1-14C]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 14CO2 release. At this point, reduction by NaB3H4 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. |
| Databáze: | OpenAIRE |
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