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Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Changes of allosteric properties resulting from modifications at the C-terminus

par Tricot, Catherine ;Schmid, Sergio;Baur, Heinz;Villeret, Vincent ;Dideberg, Otto;Haas, Didier ;Stalon, Victor
Référence European journal of biochemistry / FEBS, 221, 1, page (555-561)
Publication Publié, 1994

Article révisé par les pairs

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Résumé :

Ornithine carbamoyltransferases (OTCases) catalyse the formation of citrulline and phosphate from ornithine and carbamoylphosphate by a thermodynamically favoured reaction. In vivo, catabolic OTCase of Pseudomonas aeruginosa promotes the reverse reaction, the phosphorolysis of citrulline. Although the enzyme is assayed in vitro in the direction of citrulline synthesis, the enzyme cannot perform this reaction in vivo due to poor affinity for carbamoylphosphate and high cooperativity towards this substrate. Furthermore, the dodecameric catabolic OTCase is an allosteric enzyme; the enzyme is stimulated by nucleoside monophosphates and inhibited by polyamines (e.g. spermidine). A previous study showed that a modification of the C-terminus of the catabolic OTCase alters the homotropic cooperativity of the enzyme. We have now investigated the importance of the C-terminus for homotropic and heterotropic cooperativity by site-directed mutagenesis. Deletion of the C-terminal Ile335 residue strongly reduced cooperativity for carbamoylphosphate and sensitivity to spermidine. These properties were essentially restored when the two C-terminal amino acids (Asp334 and Ile335) were removed by deletion. However, in this variant enzyme, AMP failed to abolish carbamoylphosphate cooperativity completely, whereas the wild-type enzyme was rendered virtually non-cooperative by AMP. An extension of catabolic ornithine carbamoyltransferase by 15 amino acid residues interfered with both homotropic and heterotropic interactions and lowered the maximal velocity. All variant enzymes had the same dodecameric structure as the wild type and differed only slightly in affinity for the second substrate ornithine. A structural model of the dodecamer, at 0.3-nm resolution, suggests that the C-terminus could be involved in trimer/trimer interaction. We propose that modifications at the C-terminus alter the trimer/trimer interface and, in addition, removes the salt bridge His5-Ile335 within a monomer. These changes profoundly and indirectly modify the allosteric transition and consequently the interactions of the dodecamer with carbamoylphosphate and effectors.