Rigby D J, Radford A
Biochim Biophys Acta. 1981 Oct 13;661(2):315-22. doi: 10.1016/0005-2744(81)90020-6.
The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650,000 to 100,000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultraviolet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibtors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.
粗糙脉孢菌的嘧啶 - 3基因编码一种双功能酶，催化嘧啶生物合成途径的前两个步骤。由于该酶的不稳定性，在纯化过程中遇到了困难。该酶失去氨甲酰磷酸合成酶（二氧化碳：氨连接酶（ADP形成，氨基甲酸酯磷酸化），EC 6.3.4.16）活性，并且表观分子量从天然的650,000变为唯一可检测片段的100,000。因此，人们尝试稳定该酶，以尽量减少这些影响。弹性蛋白酶抑制剂弹性蛋白可以减少这些影响，某些缺乏一种次要蛋白酶的紫外线敏感突变菌株也有同样的效果。氨甲酰磷酸合成酶活性丧失的性质表明该酶三级结构不稳定，使用甘油可以降低这种不稳定性。甘油在该系统中也表现出蛋白酶抑制作用。虽然一系列蛋白酶抑制剂和紫外线敏感突变体的使用可以降低氨甲酰磷酸合成酶活性的衰减速率，但只有甘油可以稳定天然分子量。我们的结果支持这样的假设，即氨甲酰磷酸合成酶活性的丧失和酶分子量的变化是蛋白水解、构象转变和进一步非共价键断裂的三步序列。
