8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5 H -phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a K i of 0.59 mm. The fact that this inhibition constant was four times lower than the K m for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5 H -phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (^)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a K i of 7 mm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k 1 = 0.27 £ 10 3 m 21´s21 , k 2 = 1.8 s 21 and half-life for dissociation t 1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using ultraviolet/visible spectroscopy, and the data were consistent with the kinetic data (K = 4.2 mm). Among the other compounds tested, two potential transition state analogs, 4-carboxybutyl(1-amino-1-carboxyethyl)phosphonate (4) and 2-amino-3-hydroxy-2-methylnonadioic acid (5) were found to be competitive inhibitors with respect to l-alanine with K i of 68 mm and 80 mm, respectively.Keywords: slow-binding inhibition; enzyme inhibitor; pyridoxal-5 H -phosphate; biotin biosynthesis; 8-amino-7-oxopelargonate synthase.Biotin is an essential cofactor for carboxylases, transcarboxylases and decarboxylases in all living organisms. However, this vitamin is only biosynthesized by microorganisms and plants. The metabolic route leading to biotin has been well studied in bacteria, such as Escherichia coli and Bacillus sphaericus [1], fungi and more recently in plants [2]. It was found that the biosynthetic scheme (Scheme 1) was invariant except for the first step which varies among biotin producing organisms. Therefore the chemistry of the individual steps starting f...
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