Slow‐binding and competitive inhibition of 8‐amino‐7‐oxopelargonate synthase, a pyridoxal‐5′‐phosphate‐dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs

Abstract

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...

Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

“…Contrary to our finding of D-alanine being a substrate for the M. tuberculosis KAPA synthase, it has previously been reported to act as a competitive inhibitor for the B. sphaericus enzyme (12). Indeed, addition of D-alanine resulted in inhibition of the M. tuberculosis KAPA synthase-catalyzed reaction between L-alanine and pimeloyl-CoA.…”

“…3, A and B) with dissociation constants, K d of 1.93 Ϯ 0.28 mM and 12.49 Ϯ 1.80 mM for L-and D-alanine, respectively. These values are in agreement with those obtained for the corresponding E. coli (7) and B. sphaericus enzymes (12), which also exhibit an increased affinity for D-alanine in the presence of pimeloyl-CoA. However, in contrast to both these enzymes (7,12), addition of increasing concentrations of D-alanine to M. tuberculosis KAPA synthase in the presence of saturating concentrations of pimeloyl-CoA, resulted in the appearance of an absorption band centered at 532 nm, apart from the formation of external aldimine (Fig.…”

“…The formation of the quinonoid intermediate of M. tuberculosis KAPA synthase with D-alanine is intriguing, because such an intermediate has not been observed previously with its counterparts from E. coli (7) and B. sphaericus (12). The absence of a fast reaction phase suggests the occurrence of a longer lag period, which is most likely the time required for the conformational transitions of the pyridine ring to facilitate the abstraction of the C␣ proton of D-alanine.…”

“…KAPA synthase has been reported to show strict substrate stereospecificity such that it utilizes only L-alanine and not D-alanine (7,12), which on the contrary acts as a competitive inhibitor for the B. sphaericus enzyme (12). We therefore undertook a comparative kinetic characterization of the interaction of the enzyme with L-and D-alanine with the purpose of understanding the kinetic basis for the substrate stereospecificity.…”

7-Keto-8-Aminopelagonic Acid Synthase as a Potential Herbicide Target

“…We have performed molecular genetic dissection using reverse genetics of antisense approach to identify AtKAPAS gene encoding KAPA synthase in the pathway of biotin biosynthesis and to characterize the phenotypic consequences of loss-of-function mutations (Hwang et al, 2003;. Many researchers have investigated the KAPAS in microorganisms and the most of these reports were focused on the biosynthesis in microorganisms (Eisenberg and Star, 1968), purification and characterization (Ploux 1992, Stoner andEisenberg, 1975a;1975b), crystal structure (Alexeev et al, 1998, Kack et al, 1999, binding and kinetics (Ploux et al, 1999), point mutation (Andrew et al, 2002), and stereospecificity (Vikrant et al, 2006). Among them, Ploux et al (1999) reported that the KAPAS catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants, and suggested that the inhibitors of this pathway might lead to antifungal or herbicide agents.…”