CATABOLIC ORNITHINE CARBAMOYLTRANSFERASE OF PSEUDOMONAS-AERUGINOSA - IMPORTANCE OF THE N-TERMINAL REGION FOR DODECAMERIC STRUCTURE AND HOMOTROPIC CARBAMOYLPHOSPHATE COOPERATIVITY

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) o rnithine carbamoyltransferase (OTCase). Despite extensive sequence sim ilarities, these enzymes function unidirectionally in vivo. In the dod ecameric catabolic OTCase, homotropic cooperativity for carbamoylphosp hate strongly depresses the anabolic reaction; the residue Glu105 and the C-terminus are known to be essential for this cooperativity. When Glu105 and nine C-terminal amino acids of the catabolic OTCase were in troduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displ ayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize thes e residues, we constructed several hybrid enzymes by fusing, in vivo o r in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hy brid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were bot h trimers with little or no cooperativity. Replacing the seven N-termi nal residues of the ArcB enzyme by the corresponding six residues of E . coli ArgF enzyme produced a dodecameric enzyme which showed a reduce d affinity for carbamoylphosphate and an increase in homotropic cooper ativity, Thus, the N-terminal amino acids of catabolic OTCase are impo rtant for interaction with carbamoylphosphate, but do not alone determ ine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB res idues were both trimeric, yet they retained some homotropic cooperativ ity. Within the N-terminal ArcB region, a replacement of motif 28-33 b y the corresponding ArgF segment destabilized the dodecameric structur e and the enzyme existed in trimeric and dodecameric states, indicatin g that this region is important for dodecameric assembly. These findin gs were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer intera ctions. Dodecameric assembly appears to require at least three regions : the N- and C-termini (which are close to each other in a monomer), r esidues 28-33 and residues 147-154. Dodecameric structure correlates w ith high carbamoylphosphate cooperativity and thermal stability, but s ome trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu105-->Ala mutant gives hyperbolic carbamoylphosphate saturation, in dicating that dodecameric structure is neither necessary nor sufficien t to ensure cooperativity.