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'-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'-phosphate, in the active site of the enzyme, was studied using ultravioiet/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 Ki of 7 mu M. 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 x 10(3) M-1 . s(-1), k(2) = 1.8 s(-1) 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 mu M). 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 mu M and 80 mu M, respectively.
