Investigation of the mechanism of D-ring inversion in porphyrin biosynthesis

生理性卟啉的侧链不对称性是由羟甲基双环丙烷合酶和尿卟啉原III合酶(UROS)协同作用产生的。尽管尿素酶在卟啉生物合成化学中的作用是必不可少的，但尿素酶在结构和机械方面的许多方面仍有待研究。本课题建立了人尿路蛋白在大肠杆菌中的表达系统和纯化程序。裂解物中的酶是不稳定的，但甘油能够防止裂解物中的活性在裂解后至少几天内丧失。因此，该酶是在甘油存在的情况下提纯的，整个过程在裂解后48小时内借助高效液相色谱系统完成。纯化的酶具有显著的热稳定性，特别是在含有DTT或EDTA的磷酸盐缓冲液中，表明酶的活性可能与其氧化状态有关。对热失活过程中可获得的半胱氨酸残基数量和剩余活性之间的关系的研究表明，特定的半胱氨酸残基与活性损失有关。从人尿路的晶体结构来看，该半胱氨酸残基被认为是深埋在酶中的Cys73。Cys73-Arg突变在先天性红细胞生成性卟啉症(CEP)中最为常见，在33%的CEP患者中发现了Uros缺陷的等位基因。这些发现表明，人类尿路中的Cys73在催化中具有重要作用。从化学修饰的结果表明，几个碱性残基的作用，在推测的活性部位周围的Lys，Arg和他的残基发生了突变。突变酶的活性和动力学参数假设底物结合在两个相对的残基之间，并被另一个残基包围。

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen III synthase(UROS). Although the role of UROS is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of UROS have yet to be studied. An expression system in E.coli and a purification procedure for human UROS were established in this project. The enzyme in the lysate was unstable, but glycerol is able to prevent the activity loss in the lysate for at least a few days after lysis. The enzyme therefore was purified in the presence of glycerol, the entire procedure being completed within 48 hr after lysis with the aid of an HPLC system. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of accessible Cys residues and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human UROS, this Cys residue was considered to be Cys73, which is deeply buried inside the enzyme. A mutation of Cys73 to Arg is most common in congenital erythropoietic porphyria(CEP), in which UROS is deficient, having been found in the allele of 33% of CEP patients. These findings suggest that Cys73 of human UROS has an important role in catalysis. From the results of chemical modifications that indicate the role of several basic residues, Lys, Arg and His residues around putative active site were mutated. The activity and kinetic parameters of mutated enzymes suppose that substrate is bound between two opposite residues and surrounded by the other residues.