Biotin is an essential
enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the
biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens.
7-Keto-8-aminopelargonic acid (
KAPA) synthase (EC 2.3.1.47), the
enzyme catalyzing the first committed step in the
biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the mycobacterium tuberculosis
KAPA synthase with both L- AND D-
alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the
enzyme. The formation of the external aldimine with D-
alanine (k = 82.63 m(-1) s(-1)) is approximately 5 times slower than that with
L-alanine (k = 399.4 m(-1) s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of
pimeloyl-CoA to the preformed d-
alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with
L-alanine and
pimeloyl-CoA (k = 16.9 x 10(4) m(-1) s(-1)). Contrary to earlier reports, these results clearly show that D-
alanine is not a competitive inhibitor but a substrate for the
enzyme and thereby demonstrate the broad substrate stereospecificity of the M.
tuberculosis KAPA synthase. Further, d-KAPA, the product of the reaction utilizing D-
alanine inhibits both
KAPA synthase (Ki = 114.83 microm) as well as
7,8-diaminopelargonic acid synthase (IC50 = 43.9 microm), the next
enzyme of the pathway.