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Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis
S. Kanodia, S. Agarwal, , S. Agarwal, P. Singh, R. Bhatnagar
Published in The Biochemical Society of the Republic of Korea
PMID: 19192393
Volume: 42
Issue: 1
Pages: 47 - 52
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 Km of 3 mM for L-alanine, and a Vmax of 295 μmoles/min/mg, with the optimum activity occurring at 37°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 Ki of 160 μM and 30 mM, respectively.
About the journal
JournalBMB Reports
PublisherThe Biochemical Society of the Republic of Korea