Irvine H S, Shaw S M, Paton A, Carrey E A
Department of Biochemistry, Medical Sciences Institute, University of Dundee, Scotland.
Eur J Biochem. 1997 Aug 1;247(3):1063-73. doi: 10.1111/j.1432-1033.1997.01063.x.
Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5'-phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell. We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.
氨甲酰磷酸是氨甲酰磷酸合成酶（CPS II）活性的产物，也是天冬氨酸转氨甲酰酶（ATCase）活性的底物，这两种酶都存在于CAD中，CAD是哺乳动物中一种分子量为240 kDa的大型多酶多肽，催化嘧啶生物合成的前三个步骤。在我们对不稳定中间体在两个活性位点之间转移的研究中，我们使用了一些检测方法，这些方法能够区分氨甲酰磷酸的合成与CPS II和ATCase产生氨甲酰天冬氨酸的整体反应。我们提供了过量的外源性氨甲酰磷酸，并通过放射性标记的碳酸氢盐生成氨甲酰磷酸和氨甲酰天冬氨酸来监测其进入各自活性位点的情况。三个特征表明折叠的CPS II和ATCase结构域之间存在相互作用，导致相互的构象变化。首先，即使存在约1 mM未标记的氨甲酰磷酸，当天冬氨酸浓度较高时，ATCase会使用内源性氨甲酰磷酸来合成放射性标记的氨甲酰天冬氨酸。相反，分离的CPS II正向反应会被过量的未标记氨甲酰磷酸抑制。其次，当底物与CPS II结合时，ATCase对氨甲酰磷酸和天冬氨酸的亲和力会受到调节。第三，当存在CPS II的底物时，过渡态类似物膦酰乙酰-L-天冬氨酸对ATCase的抑制作用较弱。所有这些效应在CPS II处于活性更高的P状态时起作用，这种状态是由高浓度的ATP和镁离子诱导的，并且当5'-磷酸核糖二磷酸（变构激活剂）与低浓度的ATP同时存在时；这些是细胞内活跃生物合成过程中会遇到的条件。我们提出了一种相互变构现象，这种现象促进了多酶多肽CAD内不稳定中间体的有效转移。在这个模型中，天冬氨酸与ATCase活性位点的结合会导致CPS II配体形式活性位点的构象变化，这保护它免受其产物氨甲酰磷酸的抑制；相反，CPS II的底物会通过增加对其底物（内源性氨甲酰磷酸和天冬氨酸）的亲和力来影响ATCase的活性位点，从而阻碍外源性氨甲酰磷酸或过渡态类似物的进入。相互变构解释了多肽内酶活性紧密关联的原因，并确保氨甲酰磷酸能够有效地合成，并专门用于嘧啶生物合成的第二步。这些条件满足了细胞中代谢通道化所需的条件。
