产黄青霉硝酸还原酶。纯化及动力学机制。

Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism.

作者信息

Renosto F, Ornitz D M, Peterson D, Segel I H

出版信息

J Biol Chem. 1981 Aug 25;256(16):8616-25.

Abstract

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...

摘要

硝酸还原酶（NADPH：硝酸氧化还原酶；EC 1.6.6.1 - 3）从产黄青霉的菌丝体中纯化至表观均一。最终制剂催化NADPH依赖、FAD介导的硝酸盐还原，比活性为170 - 225单位×mg蛋白质⁻¹。凝胶过滤和甘油密度离心分别得到斯托克斯半径为6.3 nm和沉降系数s20,w为7.4。计算分子量为199,000。在十二烷基硫酸钠凝胶上，该酶呈现两条几乎相邻的染色带，对应分子量约为97,000和98,000。该酶对NADPH的偏好高于NADH（特异性常数比值kspec = 2813），对FAD的偏好高于FMN（kspec = 141），对FAD（+ NADPH）的偏好高于FADH2（kspec = 12,000），对硝酸盐的偏好高于氯酸盐（kspec = 4.33），其中kspec（给定底物的特异性常数）代表Vmax/Km。青霉属的这种酶还将催化NADPH依赖、FAD介导的细胞色素c还原，比活性为647单位×mg蛋白质⁻¹（细胞色素c的Km值Kmcyt = 1.25×10⁻⁵ M），以及还原态甲基紫精（MVH2，即甲基紫精 + 连二亚硫酸盐）依赖、NADPH和FAD非依赖的硝酸盐还原，比活性为250单位×mg蛋白质⁻¹（甲基紫精的Km值kmMVH2 = 3.5×10⁻⁶ M）。初始速度研究显示NADPH - FAD和硝酸盐 - FAD双倒数图模式相交。NADPH - 硝酸盐模式在饱和和不饱和FAD水平下是一系列平行线。NADP⁺与NADPH竞争，与硝酸盐非竞争性（在饱和和不饱和FAD水平下），对FAD是混合型抑制剂。亚硝酸盐与硝酸盐竞争，与NADPH非竞争性（在饱和和不饱和FAD水平下），对FAD是混合型抑制剂。在不饱和的硝酸盐和FAD条件下，NADPH表现出底物抑制，可能是由于与FAD位点结合。在非常低的FAD浓度下，低浓度的NADP⁺会轻微激活反应。初始速度和产物抑制模式与两种动力学机制中的任何一种都一致。一种（相当不太可能）机制涉及所有配体的快速平衡随机结合，其中（a）NADP⁺和NADPH相互排斥，（b）硝酸盐和亚硝酸盐相互排斥，（c）NADPH的结合强烈抑制硝酸盐的结合，反之亦然，（d）NADPH的结合强烈促进亚硝酸盐的结合，反之亦然，以及（e）硝酸盐的结合强烈促进NADP⁺的结合，反之亦然……