多粘芽孢杆菌中的氨同化作用。15N核磁共振及酶学研究。

Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies.

作者信息

Kanamori K, Weiss R L, Roberts J D

出版信息

J Biol Chem. 1987 Aug 15;262(23):11038-45.

Abstract

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.

摘要

通过¹⁵N核磁共振光谱法，并结合谷氨酸脱氢酶、谷氨酰胺合成酶、谷氨酸合成酶、丙氨酸脱氢酶和谷氨酰 - 丙氨酸转氨酶的比活性测定，研究了多粘芽孢杆菌中氨同化为谷氨酸和丙氨酸的途径。结果发现，不仅在以氨为氮源生长的细胞中，而且在以硝酸盐为氮源生长的细胞和固氮细胞中（其细胞内NH₄⁺浓度分别为11.2 mM、1.04 mM和1.5 mM），氨主要通过依赖NADPH的谷氨酸脱氢酶同化为谷氨酸，该酶对NH₄⁺的Km值为2.9 mM。在以氨为氮源生长的细胞中，丙氨酸脱氢酶的比活性高于谷氨酰 - 丙氨酸转氨酶，但谷氨酸脱氢酶/谷氨酰 - 丙氨酸转氨酶途径被发现是¹⁵NH₄⁺同化为[¹⁵N]丙氨酸的主要途径。将谷氨酸脱氢酶和谷氨酰胺合成酶在底物和辅酶浓度达到酶饱和时的体外比活性（分别代表谷氨酸和谷氨酰胺的合成速率）与通过核磁共振观察到的[¹⁵N]谷氨酸和[α,γ-¹⁵N]谷氨酰胺的体内生物合成速率进行了比较，并讨论了结果对限制氨和硝酸盐生长细胞中它们生物合成速率的因素的影响。