Tricot C, Schmid S, Baur H, Villeret V, Dideberg O, Haas D, Stalon V
Laboratoire de Microbiologie, Faculté des Sciences, Bruxelles, Belgique.
Eur J Biochem. 1994 Apr 1;221(1):555-61. doi: 10.1111/j.1432-1033.1994.tb18768.x.
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.
鸟氨酸氨甲酰基转移酶（OTCases）通过热力学有利反应催化鸟氨酸和氨甲酰磷酸形成瓜氨酸和磷酸。在体内，铜绿假单胞菌的分解代谢型OTCase促进反向反应，即瓜氨酸的磷酸解。尽管该酶在体外是按照瓜氨酸合成方向进行测定的，但由于对氨甲酰磷酸的亲和力低且对该底物具有高协同性，该酶在体内无法进行此反应。此外，十二聚体分解代谢型OTCase是一种别构酶；该酶受核苷单磷酸刺激并受多胺（如亚精胺）抑制。先前的一项研究表明，分解代谢型OTCase的C末端修饰会改变该酶的同促协同性。我们现在通过定点诱变研究了C末端对同促和异促协同性的重要性。C末端Ile335残基的缺失强烈降低了对氨甲酰磷酸的协同性以及对亚精胺的敏感性。当通过缺失去除两个C末端氨基酸（Asp334和Ile335）时，这些特性基本恢复。然而，在这种变体酶中，AMP未能完全消除氨甲酰磷酸的协同性，而野生型酶几乎被AMP变为非协同性。分解代谢型鸟氨酸氨甲酰基转移酶延伸15个氨基酸残基会干扰同促和异促相互作用并降低最大速度。所有变体酶都具有与野生型相同的十二聚体结构，并且对第二种底物鸟氨酸的亲和力仅略有不同。分辨率为0.3纳米的十二聚体结构模型表明，C末端可能参与三聚体/三聚体相互作用。我们提出，C末端的修饰会改变三聚体/三聚体界面，此外，还会去除单体中的盐桥His5-Ile335。这些变化深刻且间接地改变了别构转变，从而改变了十二聚体与氨甲酰磷酸和效应物的相互作用。
