Imai T
Department of Chemistry, College of General Education, Nagoya University, Aichi.
J Biochem. 1989 Nov;106(5):928-37. doi: 10.1093/oxfordjournals.jbchem.a122953.
A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.
兔脾脏中的一种颗粒状NMN糖水解酶用Triton X100增溶，并纯化了约100倍。该酶的最适pH为6.5，Km为0.25 mM，Vmax为5.3 μmol/分钟/毫克蛋白，活化能为7.9千卡/摩尔，分子量约为400,000。纯化酶和颗粒酶在底物特异性、活化能、pH曲线以及与烟酸的交换反应方面表现出相同的催化特性，只是与颗粒酶相比，纯化酶被Triton X100高度活化；似乎纯化酶具有与组织中存在的酶相同的催化特性，并且增溶不会显著改变天然蛋白质。除了对NMN具有催化活性外，兔脾脏酶还催化NAD和NADP的不可逆水解，NMN:NAD:NADP的催化活性比分别为1.00:1.45:0.44，Vmax/Km比分别为1.00:1.7:2.3。在整个酶的纯化过程中，这些活性比保持恒定，未检测到这些活性的分离。酶与Ki值类似于Km的相互竞争性抑制，以及酶的热变性速率和与NMN或NAD的活性-pH曲线相同，表明吡啶核苷酸的C-N糖苷键的水解由同一种酶催化。该酶对底物二核苷酸的嘌呤结构特异性较低，但对裂解的糖苷键具有立体特异性。烟酰胺核糖、烟酸类似物和还原形式不被水解。烟酰胺对NMN水解的线性非竞争性抑制表明是一种有序的单底物-双产物机制，其中烟酰胺是从酶中释放出的第一个产物。兔脾脏酶似乎与其他NAD糖水解酶共有的一个特性是转糖基化反应。在NMN和烟酸存在下，酶催化的转糖基化反应与水解的比率表明，该酶在体内可能作为主要的转糖基酶而非水解酶发挥作用。
