pimeloyl-coenzyme-a and 7-keto-8-aminopelargonic-acid

Over the last years, significant progress has been made in the understanding of the genetics and enzymology of the biosynthetic pathway of the vitamin biotin. The enzymes catalyzing the last four steps of this pathway, from pimeloyl-CoA to biotin, provide an ensemble of intriguing reaction mechanisms, which are presently being unravelled. The three-dimensional structures for three of these enzymes are known and provide a framework to which on-going mechanistic studies can be related.

Topics: Acyl Coenzyme A; Acyltransferases; Amino Acids; Biotin; Carbon-Nitrogen Ligases; Enzymes; Models, Molecular; Structure-Activity Relationship; Transaminases

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.

Topics: Acyl Coenzyme A; Acyltransferases; Alanine; Amino Acids; Kinetics; Mass Spectrometry; Models, Chemical; Mycobacterium tuberculosis; Spectrophotometry; Stereoisomerism; Substrate Specificity; Temperature; Thermodynamics