Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors.
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.
Laber
B
B
Schering AG, Berlin, Germany.
Gerbling
K P
KP
Harde
C
C
Neff
K H
KH
Nordhoff
E
E
Pohlenz
H D
HD
D015763:2-Amino-5-phosphonovalerate / Q000031:analogs & derivatives / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism; D019757:Carbon-Oxygen Lyases; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006714:Homoserine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000494:pharmacology; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D009943:Organophosphorus Compounds; C084331:2-amino-3-((phosphonomethyl)thio)propionic acid; C011066:O-phosphohomoserine; C006507:vinylglycine; D006714:Homoserine; D015763:2-Amino-5-phosphonovalerate; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C023160:threonine synthase; D005998:Glycine