Nakano K, Omura Y, Tagaya M, Fukui T
Institute of Scientific and Industrial Research, Osaka University.
J Biochem. 1989 Sep;106(3):528-32. doi: 10.1093/oxfordjournals.jbchem.a122886.
UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30%. The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid. Product inhibition studies revealed the sequential binding of UDP-glucose and MgPPi and the sequential release of glucose-1-phosphate and MgUTP, in this order. Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPPi and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor. The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus. For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced. The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities.
来自马铃薯块茎的UDP-葡萄糖焦磷酸化酶被纯化了243倍，达到近乎纯的状态，回收率为30%。纯化后的酶以UDP-葡萄糖而非ADP-葡萄糖作为底物，且不受3-磷酸甘油酸的激活。产物抑制研究表明，UDP-葡萄糖和MgPPi依次结合，葡萄糖-1-磷酸和MgUTP依次释放。对Mg2+对酶活性影响的分析表明，MgPPi和MgUTP复合物是酶反应的实际底物，而游离的UTP起抑制剂的作用。该酶可能以一种氨基末端封闭的约50 kDa多肽单体形式存在。为了进行结构比较，对从S-羧甲基化酶的胰蛋白酶消化物中分离出的29个肽段进行了测序。结果表明，马铃薯块茎酶与黏菌的UDP-葡萄糖焦磷酸化酶同源，但与大肠杆菌的ADP-葡萄糖焦磷酸化酶不同源，并提供了结构证据，证明UDP-葡萄糖和ADP-葡萄糖焦磷酸化酶是两种不同的蛋白质实体。
