Purification and properties of a succinyltransferase from Pseudomonas aeruginosa specific for both arginine and ornithine.

The arginine and ornithine succinyltransferase from Pseudomonas aeruginosa, a bifunctional enzyme involved in the aerobic utilization of arginine and ornithine, has been purified to homogeneity. The apparent molecular mass of the native enzyme was 150 kDa by gel filtration and 140 kDa by polyacrylamide gel electrophoresis under non-denaturing conditions. After SDS/PAGE two subunits of 35 kDa and 37 kDa were evident, indicating that the enzyme is a heterotetramer. Microsequence analysis of the electroblotted protein bands gave two different but well-conserved N-terminal amino acid sequences. The L-arginine saturation curve followed Henri-Michaelis kinetics with an apparent Km value of 0.5 mM. The sigmoidal saturation curve for L-ornithine indicated allosteric behaviour. D-Arginine, a competitive inhibitor with respect to L-arginine, reduced L-ornithine cooperativity. In the presence of spermidine, the L-ornithine saturation curve became increasingly sigmoidal, the Hill coefficient shifting from 2.5 in the absence of the inhibitor, to 3.5 in the presence of 20 mM spermidine. The L-arginine analog, L-homoarginine, was also a substrate of the succinyltransferase, and the saturation of the enzyme by this substrate was also cooperative. All these data confirmed the allosteric nature of the enzyme. Moreover, a mutant growing faster on L-ornithine than the parent strain had a modified succinyltransferase with a reduced L-ornithine cooperativity. The fate of L-homoarginine was different depending on whether the succinyltransferase was induced or not; excreted succinylhomoarginine was found in cultures induced for the transferase activity whereas guanidinovalerate was excreted in non-induced cultures. The 'waste' of succinyl CoA, which could not be regenerated from the excreted succinylhomoarginine, explained the inhibition exerted by L-homoarginine on growth when ornithine or arginine was used as the growth medium.