ALANINE RACEMASE FROM AN ACIDOPHILE, ACIDIPHILIUM-ORGANOVORUM - PURIFICATION AND CHARACTERIZATION

An alanine racemase (EC 5.1.1.1) from an acidophilic heterotrophic bac terium, Acidiphilium organovorum 13H, was purified and characterized. The enzyme had a dimeric structure with identical subunits of M-r 33,0 00 each. Although A. organovorum 13H is an acidophile, the enzyme had its maximum velocity at pH 9, corresponding to its location in the cyt oplasm. Activity was maximum between 50 and 60 degrees C. For an enzym e from a mesophile, it was stable to heat, showing no loss of activity after a 30-min incubation at 65 degrees C. The enzyme needed pyridoxa l 5'-phosphate (PLP) as a cofactor for its activity, as seen from the loss of activity upon dialysis against PLP-free buffer containing hydr oxylamine and its absorption maximum at 420 nm. Activity was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content studies fo und that 1 mole of enzyme contained 2 moles of PLP. The enzyme catalyz ed the symmetric reversible racemization of alanine exclusively.