Guanosine-inosine-preferring nucleoside N-glycohydrolase from Crithidia fasciculata.
Guanosine-inosine-preferring nucleoside N-glycohydrolase from Crithidia fasciculata.
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发表时间：
1994-09
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通讯作者：
B. Estupiñán;V. Schramm
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作者：
B. Estupiñán;V. Schramm
原生动物寄生虫无法从头生物合成嘌呤并通过补救途径获得嘌呤。偏好肌苷和尿苷作为底物的核苷水解酶（IU-核苷水解酶）已被表征并涉及 Crithidia fasciculata 中的嘌呤补救（Parkin, D. W.、Horenstein, B. A.、Abdulah, D. R.、Estupiñán, B. 和 Schramm, V. L. (1991) J. Biol. Chem. 31， 20658-20665）。用 IU-核苷水解酶抑制剂处理 C. fasciculata 并不能阻止细胞生长，表明有替代酶。一种鸟苷-肌苷偏爱酶（GI-核苷水解酶）已从 C. fasciculata 提取物中纯化出来并进行了表征。该酶是 M(r) 38,500 个亚基的寡聚物。鸟苷、肌苷和腺苷的 Vmax/Km 分别为 3.2 x 10(6)、6.2 x 10(6) 和 9.8 M-1 S-1。脱氧核苷、核苷酸和嘧啶核苷是不良底物。 Km 的 pH 曲线与 pH 无关，而 Vmax 和 Vmax/Km 均表明活性需要单一质子化碱基 pKa 7.7。 IU-核苷水解酶、1，4-二脱氧-1，4-亚氨基-1-(S)-苯基-D-核糖醇的过渡态抑制剂(Horenstein，B.A.和Schramm，V.L.(1993)Biochemistry 32， 9917-9925)和对硝基苯基核糖腙(Boutellier，M.，Horenstein，B.A.， Semenyaka, A.、Schramm, V.L. 和 Ganem, B. (1994) Biochemistry 33, 3994-4000) 是胃肠道核苷水解酶的普通抑制剂。该酶被 3-脱氮腺苷和 2-碘腺苷抑制，Km/Ki 值分别为 145 和 61。结果表明，这种以前未表征的酶相对于 IU-核苷水解酶具有独特的结构、动力学和化学机制。以标记肌苷作为唯一嘌呤来源的代谢研究表明，胃肠道酶对于体内嘌呤回收是有效的。
Protozoan parasites are incapable of de novo purine biosynthesis and obtain purines by salvage pathways. A nucleoside hydrolase which prefers inosine and uridine as substrates (IU-nucleoside hydrolase) has been characterized and implicated in purine salvage in Crithidia fasciculata (Parkin, D. W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., and Schramm, V. L. (1991) J. Biol. Chem. 31, 20658-20665). Treatment of C. fasciculata with inhibitors of the IU-nucleoside hydrolase did not prevent cell growth, suggesting alternative enzymes. A guanosine-inosine-preferring enzyme (GI-nucleoside hydrolase) has been purified from extracts of C. fasciculata and characterized. The enzyme is an oligomer of M(r) 38,500 subunits. The Vmax/Km for guanosine, inosine, and adenosine are 3.2 x 10(6), 6.2 x 10(6), and 9.8 M-1 S-1, respectively. Deoxynucleosides, nucleotides, and pyrimidine nucleosides are poor substrates. The pH profile for Km is independent of pH, whereas both Vmax and Vmax/Km demonstrate that a single protonated base, pKa 7.7 is required for activity. The transition state inhibitors of IU-nucleoside hydrolase, 1,4-dideoxy-1,4-imino-1-(S)-phenyl-D-ribitol (Horenstein, B. A., and Schramm, V. L. (1993) Biochemistry 32, 9917-9925) and p-nitrophenylriboamidrazone (Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V.L., and Ganem, B. (1994) Biochemistry 33, 3994-4000), are unexceptional inhibitors of the GI-nucleoside hydrolase. The enzyme is inhibited by 3-deazaadenosine and 2-iodoadenosine with Km/Ki values of 145 and 61, respectively. The results demonstrate that this previously uncharacterized enzyme has distinct structure, kinetic, and chemical mechanisms relative to IU-nucleoside hydrolase. Metabolic studies with labeled inosine as the sole purine source indicated that the GI-enzyme is efficient for purine salvage in vivo.