Norberg P, Kaplan J G, Kushner D J
J Bacteriol. 1973 Feb;113(2):680-6. doi: 10.1128/jb.113.2.680-686.1973.
Properties of the aspartate transcarbamylase of the extremly halophilic bacterium Halobacterium cutirubrum, an enzyme that needs high salt concentrations for activity and regulation, were studied in cell-free extracts. The enzyme was stable on prolonged incubation at 4 C in concentrated extracts (50 mg of protein per ml) but not in diluted extracts. Mg(2+) ions and beta-mercaptoethanol stabilized enzyme activity. At salt concentrations below the maximum for activity (3.5 m), the enzyme was rapidly inactivated. Carbamyl phosphate stabilized the enzyme under these conditions; aspartate had a smaller effect. The enzyme was most stable at 0 C; raising or lowering the temperature from this point increased the rate of inactivation. On exposure to lowered salt concentrations, enzyme activity was more sensitive than feedback inhibition. Hyperbolic substrate saturation curves were found for carbamyl phosphate. The K(m) obtained varied with the salt concentration used. With aspartate, sigmoidal curves were found when extracts were assayed immediately after preparation, but hyperbolic curves were obtained with extracts allowed to stand 1 to 2 hr. The presence of cytidine triphosphate (CTP) decreased the V(max) but did not change the K(m); this is thus a V-type enzyme. Low concentrations of succinate activated the enzyme, in the presence and absence of CTP; higher concentrations did not affect its activity. CTP increased the activation energy of the enzyme in 3.5 m salt but decreased it in 2.0 m salt. At both salt concentrations, the sensitivity of the enzyme to feedback inhibition diminished with increasing temperatures. Gel chromatography suggested that the enzyme in crude extracts had a molecular weight of 160,000. Precipitating the enzyme with polyethylene glycol decreased the molecular weight to 34,000, and this activity was no longer sensitive to CTP. The presence of either substrate of the enzyme during polyethylene glycol treatment prevented dissociation of the enzyme and loss of feedback inhibition. Thus, as with other aspartate transcarbamylases, association of subunits seems to be required for regulation of activity by end product.
对极端嗜盐细菌红皮盐杆菌的天冬氨酸转氨甲酰酶的性质进行了研究，该酶需要高盐浓度来进行活性调节，研究是在无细胞提取物中进行的。该酶在浓缩提取物（每毫升50毫克蛋白质）中于4℃长时间孵育时稳定，但在稀释提取物中不稳定。镁离子和β-巯基乙醇可稳定酶活性。在盐浓度低于活性最大值（3.5m）时，酶会迅速失活。在这些条件下，氨甲酰磷酸可稳定酶；天冬氨酸的作用较小。该酶在0℃时最稳定；从这一温度升高或降低温度都会增加失活速率。暴露于低盐浓度时，酶活性比反馈抑制更敏感。发现氨甲酰磷酸的底物饱和曲线为双曲线。获得的K(m)随所用盐浓度而变化。对于天冬氨酸，制备后立即测定提取物时发现为S形曲线，但提取物静置1至2小时后获得的是双曲线。胞苷三磷酸（CTP）的存在降低了V(max)但未改变K(m)；因此这是一种V型酶。低浓度的琥珀酸在有和没有CTP的情况下均激活该酶；较高浓度则不影响其活性。CTP在3.5m盐中增加了酶的活化能，但在2.0m盐中降低了活化能。在这两种盐浓度下，酶对反馈抑制的敏感性均随温度升高而降低。凝胶色谱表明粗提取物中的酶分子量为160,000。用聚乙二醇沉淀酶可使分子量降至34,000，且该活性不再对CTP敏感。聚乙二醇处理期间酶的任何一种底物的存在可防止酶解离和反馈抑制丧失。因此，与其他天冬氨酸转氨甲酰酶一样，亚基的缔合似乎是终产物调节活性所必需的。
