Hinde R W, Jacobson J A, Weiss R L, Davis R H
J Biol Chem. 1986 May 5;261(13):5848-52.
N-Acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." Because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. We have detected this enzyme in crude extracts of Neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. The enzyme is bound to the mitochondrial membrane. The enzyme has a pH optimum of 9.0 and Km values for glutamate and CoASAc of 6.3 and 1.6 mM, respectively. It is feedback-inhibited by L-arginine (I0.5 = 0.16 mM), and its specific activity is augmented 2-3-fold by arginine starvation of the mycelium. Mutants of the newly recognized arg-14 locus lack activity for the enzyme. Because these mutants are complete auxotrophs, we conclude that N-acetylglutamate synthase is an indispensible enzyme of arginine biosynthesis in N. crassa. This work completes the assignment of enzymes of the arginine pathway of N. crassa to corresponding genetic loci. The membrane localization of the enzyme suggests a novel mechanism by which feedback inhibition might occur across a semipermeable membrane.
N-乙酰谷氨酸合酶是精氨酸途径的一种早期酶，在所谓的“乙酰谷氨酸循环”中为鸟氨酸合成提供乙酰谷氨酸。由于在鸟氨酸形成时乙酰谷氨酸会再生，该酶在该途径中仅具有催化或补充作用，在生长过程中维持“结合”的乙酰基团。我们在粗糙脉孢菌的粗提物中检测到了这种酶，并将其与其他鸟氨酸生物合成酶一起定位到线粒体中。该酶与线粒体外膜结合。该酶的最适pH为9.0，谷氨酸和乙酰辅酶A的Km值分别为6.3 mM和1.6 mM。它受到L-精氨酸的反馈抑制（I0.5 = 0.16 mM），菌丝体经精氨酸饥饿处理后其比活性会增加2至3倍。新发现的arg-14位点的突变体缺乏该酶的活性。由于这些突变体是完全营养缺陷型，我们得出结论，N-乙酰谷氨酸合酶是粗糙脉孢菌中精氨酸生物合成所必需的酶。这项工作完成了将粗糙脉孢菌精氨酸途径的酶分配到相应基因位点的任务。该酶的膜定位提示了一种反馈抑制可能通过半透膜发生的新机制。
