PURIFICATION AND PROPERTIES OF A SUCCINYLTRANSFERASE FROM PSEUDOMONAS-AERUGINOSA SPECIFIC FOR BOTH ARGININE AND ORNITHINE

The arginine and ornithine succinyltransferase from Pseudomonas aerugi nosa, a bifunctional enzyme involved in the aerobic utilization of arg inine and ornithine, has been purified to homogeneity. The apparent mo lecular mass of the native enzyme was 150 kDa by gel filtration and 14 0 kDa by polyacrylamide gel electrophoresis under non-denaturing condi tions. 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-conse rved N-terminal amino acid sequences. The L-arginine saturation curve followed Henri-Michaelis kinetics with an apparent K, value of 0.5 mM. The sigmoidal saturation curve for L-ornithine indicated allosteric b ehaviour. D-Arginine, a competitive inhibitor with respect to L-argini ne, reduced L-ornithine cooperativity. In the presence of spermidine, the L-ornithine saturation curve became increasingly sigmoidal, the Hi ll 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-homoa rginine, was also a substrate of the succinyltransferase, and the satu ration of the enzyme by this substrate was also cooperative. All these data confirmed the allosteric nature of the enzyme. Moreover, a mutan t 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 succinyltran sferase was induced or not; excreted succinylhomoarginine was found in cultures induced for the transferase activity whereas guanidinovalera te 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 orn ithine or arginine was used as the growth medium.