Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors.
Biochemistry
; 33(11): 3413-23, 1994 Mar 22.
Article
em En
| MEDLINE
| ID: mdl-7907888
Threonine synthase (TS), the last enzyme of the threonine biosynthetic pathway, catalyzes L-threonine formation from L-homoserine phosphate (HSerP; Km = 0.5 mM, V = 440 min-1) and DL-vinylglycine. Furthermore, TS catalyzes beta-elimination reactions with L-serine (Km = 150 mM, V = 4.7 min-1), DL-3-chloroalanine, L-threonine, and L-allo-threonine as substrates to yield pyruvate or alpha-ketobutyrate, while L-alanine, L-2-aminobutanoic acid, and L-2-amino-5-phosphonopentanoic acid are substrates for half-transamination reactions to form the pyridoxamine form of the enzyme and the corresponding alpha-keto acid. Spectral analyses of all these reactions revealed the transient formation of strongly absorbing long-wavelength chromophores (lambda max = 440-445 nm), implying the accumulation of the corresponding pyridoxaldimine p-quinonoidal intermediates. HSerP turnover was competitively inhibited by L-3-hydroxyhomoserine phosphate 1 (Ki = 0.050 mM), L-2,3-methanohomoserine phosphate 2 (Ki = 0.010 mM), L-2-amino-3-[(phosphonomethyl)thio)]propanoic acid 5 (Ki = 0.011 mM) and DL-E-2-amino-5-phosphono-4-pentenoic acid 10 (Ki = 0.54 mM). 5 and 10 induced the formation of long-wavelength quinonoidal chromophores (lambda max = 458 and 460 mm, epsilon 47,000 and 30,000 M-1 cm-1), while incubation with either 1 or 2 induced only minor spectral changes. DL-2-Amino-3-[(phosphonomethyl)amino)]propanoic acid inactivated TS (Ki = 0.057 mM, kinact = 1.44 min-1) with 1:1 stoichiometry, transient formation of a 450-nm chromophore, and finally bleaching of any absorbance at wavelengths longer than 320 nm. Z-2-Amino-5-phosphono-3-pentenoic acid 8 is the unusual amino acid found in the peptide antibiotics of the plumbemicin and rhizocticin families. Racemic 8 irreversibly inhibited TS (Ki = 0.1 mM, kinact = 1.50 min-1) with 1:1 stoichiometry and the concomitant formation of a 482-nm chromophore (epsilon approximately 30,000 M-1 cm-1). DL-E-2-Amino-5-phosphono-3-pentenoic acid was a less potent irreversible inhibitor of TS (Ki = 0.4 mM, kinact = 0.25 min-1), inducing absorption maxima at 462 and 500 nm. The acetylenic amino acid DL-2-amino-5-phosphono-4-pentynoic acid 12 bound to TS (KD = 0.38 mM) forming a quinonoidal chromophore (lambda max = 452 nm, epsilon approximately 30,000 M-1 cm-1), but inhibition of the enzyme by 12 could not be detected under assay conditions even at high inhibitor concentrations. Mechanisms consistent with these observations are proposed.
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Base de dados:
MEDLINE
Assunto principal:
Carbono-Oxigênio Liases
/
Escherichia coli
/
Liases
Idioma:
En
Ano de publicação:
1994
Tipo de documento:
Article