UDP-galactose 4-epimerase: NAD +相关内容和电荷转移乐队substrate-induced构象的转变。
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刘Y, Vanhooke杰弗雷PA
UDP-galactose 4-epimerase: NAD +相关内容和电荷转移乐队substrate-induced构象的转变。
生物化学,1996年6月11日,35 (23):7615 - 20。
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8652544 (在PubMed]
- 文摘
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UDP-galactose 4-epimerase从大肠杆菌包含紧密地绑定NAD +,而参与催化UDP-galactose互变现象和UDP-glucose通过其氧化还原性质。纯化酶二聚体相同的子单元,由一个混合物催化地活跃的子单元指定E。NAD +不活跃,流产复合物指定E.NADH。尿苷核苷酸,尿苷核苷酸可能UDP-glucose, UDP-galactose或UDP (Vanhooke, j·L。&弗雷,p . a(1994)生物。化学,269,31496 - 31404]。流产复合物转化为活跃E。NAD +纯化酶的变性在6米4摄氏度盐酸胍缓冲在pH值为7.0的0.126毫米NAD + 3 h,其次是稀释的盐酸胍0.18米和0.076毫米的NAD + 2 h。用酶完全活跃和包含大量的NADH和尿苷核苷酸可以忽略不计。差向异构酶在280 nm的消光系数是1.81 + / - 0.15毫升mg-1 cm - 1(ε280 = 137 + / - 11 mm - 1 cm - 1),由氨基酸定量分析和光谱光度测量的测量。这个值允许的价值降低了酶的消光系数(E.NADH)计算ε344 = 5.7 mm - 1 cm - 1。ε的新价值280的基础上,分析测量差向异构酶的nAD +内容显示有两种分子的nAD + /二聚体,这证实了结论分析决定早些时候从x射线晶体学和修正。紫外/可见吸收光谱的E。NAD+ from denaturation-renaturation experiments reveals the presence of a broad absorption band extending from 300 nm to beyond 360 nm that cannot be attributed to NADH and appears to be a charge-transfer band. This band is partially bleached by UMP and almost totally abolished by UDP, indicating that the interactions leading to the charge-transfer band are altered by the uridine nucleotide-induced conformational change in this enzyme. This conformational change is associated with control of the chemical reactivity of NAD+ in the reaction mechanism.