Piérard A, Schröter B
J Bacteriol. 1978 Apr;134(1):167-76. doi: 10.1128/jb.134.1.167-176.1978.
The arginine pathway carbamoylphosphate synthase (CPSase A) from Saccharomyces cerevisiae was shown to be highly unstable and could not be substantially purified. In spite of this instability, a number of important properties of this enzyme were determined with crude preparations. A molecular weight of 140,000 (7.9S) was estimated for the native enzyme by sucrose gradient centrifugation; a significantly higher value, 175,000, was obtained by gel filtration on Sephadex. The enzyme is an aggregate consisting of two protein components, coded for by the unlinked genes cpaI and cpaII. These components were separated by diethylaminoethyl-cellulose chromatography. Their molecular weights, estimated by Sephadex gel filtration, were 36,000 and 130,000. The large component catalyzed the synthesis of carbamoylphosphate from ammonia. The small component was required in addition to the large one for the physiologically functional glutamine-dependent activity. Apparent Michaelis constants at pH 7.5 of 1.25 mM for glutamine and 75 mM for NH(4)Cl were measured with the native enzyme. The use of various glutamine analogs, including 2-amino-4-oxo-5-chloropentanoic acid, indicated that binding of glutamine to a site located on the small component was followed by transfer of its amide nitrogen to the ammonia site on the heavy component. This ammonia site was able to function independently of the utilization of glutamine. However, binding of glutamine was conjectured to cause a conformational change in the heavy component that greatly increased the rate of synthesis of carbamoylphosphate from ammonia. Glutamine, which was also shown to stabilize the aggregation of the two components, appeared to be a major effector of the catalytic and structural properties of CPSase A. In view of these observations, the CPSase A of yeast appears to share a number of structural and catalytic properties with the Escherichia coli enzyme. Obviously, the unlinked cpaI and cpaII genes of yeast are homologous to the adjacent carA and carB genes that code for the two subunits of the bacterial enzyme.
已证明来自酿酒酵母的精氨酸途径氨甲酰磷酸合成酶（CPSase A）极不稳定，无法大量纯化。尽管存在这种不稳定性，但仍用粗制制剂测定了该酶的一些重要特性。通过蔗糖梯度离心法估计天然酶的分子量为140,000（7.9S）；通过在葡聚糖凝胶上进行凝胶过滤得到的值明显更高，为175,000。该酶是由两个蛋白质组分组成的聚集体，由不连锁的基因cpaI和cpaII编码。这些组分通过二乙氨基乙基纤维素色谱法分离。通过葡聚糖凝胶过滤估计它们的分子量分别为36,000和130,000。较大的组分催化从氨合成氨甲酰磷酸。除了大组分外，还需要小组分来实现生理功能上依赖谷氨酰胺的活性。用天然酶在pH 7.5下测得谷氨酰胺的表观米氏常数为1.25 mM，氯化铵的表观米氏常数为75 mM。使用包括2-氨基-4-氧代-5-氯戊酸在内的各种谷氨酰胺类似物表明，谷氨酰胺与位于小组分上的位点结合后，其酰胺氮会转移到重组分上的氨位点。这个氨位点能够独立于谷氨酰胺的利用而发挥作用。然而，推测谷氨酰胺的结合会导致重组分发生构象变化，从而大大提高从氨合成氨甲酰磷酸的速率。谷氨酰胺还被证明能稳定两个组分的聚集，似乎是CPSase A催化和结构特性的主要效应物。鉴于这些观察结果，酵母的CPSase A似乎与大肠杆菌的酶具有许多结构和催化特性。显然，酵母中不连锁的cpaI和cpaII基因与编码细菌酶两个亚基的相邻carA和carB基因是同源的。
