PURIFICATION AND PROPERTIES OF UROPORPHYRINOGEN-III SYNTHASE (CO-SYNTHASE) FROM AN OVERPRODUCING RECOMBINANT STRAIN OF ESCHERICHIA-COLI K-12
PURIFICATION AND PROPERTIES OF UROPORPHYRINOGEN-III SYNTHASE (CO-SYNTHASE) FROM AN OVERPRODUCING RECOMBINANT STRAIN OF ESCHERICHIA-COLI K-12
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DOI：
10.1042/bj2640397
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发表时间：
1989-12-01
影响因子：
4.1
通讯作者：
JORDAN, PM
中科院分区：
文献类型：
--
作者：
ALWAN, AF;MGBEJE, BIA;JORDAN, PM
大肠杆菌hemD基因编码尿卟啉原III合成酶（共合成酶），该基因被克隆到大肠杆菌细胞的多拷贝质粒中，该质粒用于产生产生高达野生型合成酶浓度1000倍的菌株。酶从这些菌株中以毫克量纯化到均匀性。该酶是Mr 28000的单体，等电点为5.2，最适pH值为7.8。纯化的合成酶的比活性为1500单位/毫克，底物前尿卟啉原的Km为5 μ m。该酶的n端序列为Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-，与基因源性蛋白序列一致。该酶含有四个5,5' -二硫代（2-硝基苯甲酸）可滴定基团，其中一个与试剂反应迅速，另外三个反应活性较低。这种酶是热敏的，在热失活过程中，所有四个巯基都同样可用于反应。
The Escherichia coli hemD gene, encoding the enzyme uroporphyrinogen III synthase (co-synthase), was cloned into multi-copy plasmids in E. coli cells that were used to generate strains producing up to 1000 times the concentration of the synthase in the wild-type. The enzyme was purified to homogeneity from these strains in milligram amounts. The enzyme is a monomer of Mr 28 000 with an isoelectric point of 5.2 and a pH optimum of 7.8. The specific activity of the purified synthase is 1500 units/mg and the Km for the substrate, pre-uroporphyrinogen, is 5 .mu.M. The N-terminal sequence of the enzyme is Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-, in agreement with the gene-derived protein sequence. The enzyme contains four 5,5''-dithiobis(2-nitrobenzoic acid)-titratable groups, one reacting rapidly with the reagent and three further groups having lower reactivity. The enzyme is heat-sensitive, and during heat inactivation all four thiol groups become equally available for reaction.