Rusche K M, Spiering M M, Marletta M A
Department of Biological Chemistry, School of Medicine, Interdepartmental Program in Medicinal Chemistry, The University of Michigan, Ann Arbor 48109-1065, USA.
Biochemistry. 1998 Nov 3;37(44):15503-12. doi: 10.1021/bi9813936.
Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.
小鼠巨噬细胞一氧化氮合酶（NOS）在大肠杆菌中表达，并在存在（全酶 NOS）或不存在（无 H4B 的 NOS）（6R）-四氢-L-生物蝶呤（H4B）的情况下进行纯化。活性酶的分离需要钙调蛋白的共表达。重组全酶 NOS 表现出与从小鼠巨噬细胞分离的酶相似的光谱特征和活性。无 H4B 的 NOS 在约 420nm 处显示一个 Soret 带，通过分析凝胶过滤，由单体和二聚体的混合物组成。无 H4B 的 NOS 以过氧化氢（H2O2）或 NADPH 和 O2 为底物催化 NG-羟基-L-精氨酸（NHA）的氧化。在任何一种条件下均未观察到精氨酸形成产物。在 H2O2 和 NADPH/O2 反应中，NHA 氧化的氨基酸产物被确定为瓜氨酸和 Nδ-氰基鸟氨酸（CN-鸟氨酸）。还形成了亚硝酸盐和硝酸盐。化学发光分析未检测到 NADPH/O2 反应中一氧化氮（NO）的形成。基于使用可溶性鸟苷酸环化酶的血红素结构域作为捕获剂形成亚铁亚硝酰复合物，以及在 NHA 和 NADPH 周转期间无 H4B 的 NOS 形成亚铁亚硝酰复合物，NADPH/O2 反应的初始无机产物被认为是硝酰阴离子（NO-）。NO-不稳定，在反应条件下被氧化为亚硝酸盐和硝酸盐。在 25℃时，H2O2 支持的反应的比活性为 120±14nmol min-1 mg-1，NADPH 支持的反应的比活性为 31±6nmol min-1 mg-1，NHA 的表观 KM 为 129±9μM。全酶 NOS 催化 H2O2 支持的反应，比活性为 815±30nmol min-1 mg-1，NADPH 依赖性反应以 171±20nmol min-1 mg-1 的速率产生NO 和瓜氨酸，NADPH 反应中 NHA 的表观 KM 为 36.9±0.3μM。
