The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optim u m was 37-40 ° C, the optimum pH around 8.0 and optimal activity required 50-100 mM TRIS-HC1 buffer, p H 8.0 and 3-7 mM MgC12; MgC12 in excess of 10 mM was inhibitory. The activation energy for benzoate was l l . 3 k c a l / m o l . Although growth occurred only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3-and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The Km values for benzoate, 2-, 3-and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 m ~, the Vma× values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a Km of 0.07 mM and a Vm~, of 1.05 U/mg and for A T P a K~ of 0.16 mM and a Vm,x of 1.08 U/mg was determined.
Benzoate activation was inhibited by more than 6 m ~ ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (Ki) for pyrophosphate was 5.7 raM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.