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

生理卟啉的侧链不对称是由羟甲基二烷合成酶和尿卟啉原III合成酶（UROS）共同作用产生的。虽然UROS在卟啉生物合成的化学过程中起着至关重要的作用，但其在结构和力学方面的许多方面还有待研究。本课题建立了人UROS在大肠杆菌中的表达体系和纯化方法。裂解物中的酶是不稳定的，但甘油能够在裂解后至少几天内防止裂解物中的活性损失。因此，在甘油的存在下纯化酶，在高效液相色谱系统的帮助下，裂解后48小时内完成整个过程。纯化后的酶表现出显著的热稳定性，特别是在含有DTT或EDTA的磷酸盐缓冲液中保存时，表明酶的活性可能取决于其氧化态。对热失活过程中可接近的Cys残基数量与剩余活性之间关系的研究表明，一种特定的Cys残基参与了活性损失。从人类UROS的晶体结构来看，这个Cys残基被认为是Cys73，它深埋在酶内部。Cys73到Arg的突变在先天性红细胞生成性卟啉症（CEP）中最常见，其中UROS缺乏，在33%的CEP患者中发现了该等位基因。这些结果表明，Cys73在人UROS中具有重要的催化作用。从化学修饰的结果表明，几个基本残基的作用，赖氨酸，精氨酸和他的残基周围的推定活性位点发生突变。突变酶的活性和动力学参数假设底物结合在两个相反的残基之间，并被其他残基包围。

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.