Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.
Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.
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DOI：
10.1016/s0021-9258(18)54759-1
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发表时间：
1991-11
期刊：
影响因子：
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通讯作者：
D. Parkin;B. Horenstein;D. Abdulah;B. Estupiñán;V. Schramm
中科院分区：
文献类型：
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作者：
D. Parkin;B. Horenstein;D. Abdulah;B. Estupiñán;V. Schramm
在添加有[8- 14 C，5 '-3H]次黄嘌呤核苷或[8- 14 C，5'-3H]腺苷的复合培养基上生长的束状Crithidia fasciculata细胞通过涉及N-糖苷键裂解的途径代谢大于50%的挽救的核苷。细胞提取物含有大量的核苷水解酶活性，但不显著的嘌呤核苷磷酸化酶。该核苷水解酶已从千克量的C纯化1000倍至大于99%的均一性。束状。该酶是Mr为34，000亚基的四聚体，通过凝胶过滤得到表观全酶Mr为143，000。所有常见的核苷都是底物。Km值从0.38到4.7 mM变化，嘌呤核苷比嘧啶结合更紧密。Vmax/Km值在3.4 x 10（3）M-1 s-1至1.7 x 10（5）M-1 s-1之间变化，嘧啶核苷的值较大。在30 ℃时肌苷的周转率为32 s-1。以肌苷为底物的动力学机制为快速平衡，产物随机释放。水解反应可以逆转，得到106 M的实验Keq，H2O作为单位。核糖和次黄嘌呤的产物解离常数分别为0.7和6.2 mM。脱氧核苷或5 '-取代的核苷是不良底物或不反应，并且是酶的不良抑制剂。该酶在稳态催化过程中对甲醇的攻击有区别，表明酶介导的水亲核试剂的参与。肌苷水解的pH曲线给出两个表观pKa值6.1，其中Vmax/Km值低于pKa降低，并且在较高pH值下达到平台。这些影响是由于pH值的Vmax值的敏感性，因为Km是独立的pH值。pH值的配置文件涉及两个带负电荷的基团，稳定过渡态与氧碳正离子字符。
Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.