Bhor, Vikrant M and Dev, Sagarika and Vasanthakumar, Ganga Ramu and Kumar, Parimal and Sinha, Sharmistha and Surolia, Avadhesha (2006) Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase-Spectroscopic And Kinetic Studies. In: Journal Of Biological Chemistry, 281 (35). pp. 25076-25088.
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Abstract
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 similar to 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 (K-i = 114.83 mu M) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 mu M), the next enzyme of the pathway
Item Type: | Journal Article |
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Publication: | Journal Of Biological Chemistry |
Publisher: | American Soc Biochemistry Molecular Biology Inc |
Additional Information: | Copyright of this article belongs to American Society of Biochemistry Molecular Biology Inc. |
Keywords: | Biochemistry & Molecular Biology;8-Amino-7-Oxononanoate Synthase; Biotin Biosynthesis; 8-Amino-7-Oxopelargonate Synthase; Crystal-Structure; Enzyme; Binding;Intermediate; Specificity; Inhibition; Mechanism |
Department/Centre: | Division of Biological Sciences > Molecular Reproduction, Development & Genetics |
Date Deposited: | 10 Dec 2008 11:00 |
Last Modified: | 19 Sep 2010 04:54 |
URI: | http://eprints.iisc.ac.in/id/eprint/16919 |
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