Biochemical characterization of alanine racemase--a spore protein produced by Bacillus anthracis.
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 micromoles/min/mg, with the optimum activity occurring at 37 degrees 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 microM and 30 mM, respectively. [BMB reports 2009; 42(1): 47-52].
Kanodia
Shivani
S
Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India.
Agarwal
Shivangi
S
Singh
Priyanka
P
Agarwal
Shivani
S
Singh
Preeti
P
Bhatnagar
Rakesh
R
D000411:Alanine Racemase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001408:Bacillus anthracis / Q000187:drug effects / Q000201:enzymology*; D055162:Biocatalysis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013171:Spores, Bacterial / Q000187:drug effects / Q000201:enzymology*; D013379:Substrate Specificity / Q000187:drug effects
D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000411:Alanine Racemase