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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

  • Kanodia, Shivani (Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University) ;
  • Agarwal, Shivangi (Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University) ;
  • Singh, Priyanka (Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University) ;
  • Agarwal, Shivani (Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University) ;
  • Singh, Preeti (Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University) ;
  • Bhatnagar, Rakesh (Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University)
  • Published : 2009.01.31
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Abstract

Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent $K_m$ of 3 mM for L-alanine, and a $V_{max}$ of $295\;{\mu}moles/min/mg$, with the optimum activity occurring at $37^{\circ}C$ and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a $K_i$of $160\;{\mu}M$ and 30 mM, respectively.

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References

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