Allosteric regulation in Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase revisited: association of concerted homotropic cooperative interactions and local heterotropic effects.

The allosteric catabolic ornithine carbamoyltransferase (OTCase) from Pseudomonas aeruginosa, a dodecamer build up of four trimers of identical subunits, shows strong carbamoylphosphate homotropic co-operativity. Its activity is allosterically inhibited by spermidine and activated by AMP. Modified forms of the enzyme exhibiting substantial alterations in both homotropic and heterotropic interactions were recently obtained. We report here the first detailed kinetic characterization of homotropic and heterotropic modulations in allosteric wild-type and in engineered OTCases. Homotropic co-operativity for the saturation either by citrulline or arsenate was also observed when arsenate was utilised as an alternate substrate of the reverse reaction. Amino acid substitution of glutamate 105 by a glycine produces an enzyme devoid of homotropic interactions between the catalytic sites for carbamoylphosphate. This mutant, which is blocked in an active conformation, is still sensitive to the allosteric effector AMP, which increases affinity with respect to the substrate, carbamoylphosphate. It is also observed that homotropic co-operative interactions do not reappear in the E105G enzyme upon strong inhibition by the allosteric inhibitor of the wild-type enzyme, spermidine.Replacement of residues 34 to 101 of the native enzyme by the homologous amino acids of anabolic Escherichia coli OTCase produces a trimeric enzyme which retains reduced homotropic co-operativity. Activation by AMP and inhibition by spermidine of this chimaeric OTCase do not affect carbamoylphosphate homotropic co-operativity. AMP acts by reducing the concentration of substrate at half maximum velocity while spermidine acts in the inverse way. These observations indicate that in the two mutant forms of OTCase, homotropic and heterotropic interactions can be uncoupled and therefore must involve different molecular mechanisms. Furthermore, the results of stimulation of enzyme activity by phosphate, arsenate, pyrophosphate and phosphonoacetyl-l-ornithine on wild-type and mutant OTCases suggest that the physiological substrate phosphate, besides acting at the catalytic site, may act at an allosteric site. On the other hand, pyrophosphate and phosphonoacetyl-l-ornithine activation results exclusively from interactions of this effector with the active site residues.