大肠杆菌的系统守恒的组氨酸胆色素原合成酶催化的不需要。
文章的细节
-
引用
复制到剪贴板 -
米切尔LW,小提琴,杰夫艾克
大肠杆菌的系统守恒的组氨酸胆色素原合成酶催化的不需要。
生物化学杂志。1995年10月13日;270 (41):24054 - 9。
- PubMed ID
-
7592604 (在PubMed]
- 文摘
-
胆色素原合成酶(pbg)是一种金属酶,催化四吡咯生物合成的常见的第一步,两个分子的非对称凝结5-aminolevulinic酸(ALA)形成胆色素原。化学改性的数据表明组氨酸的催化残百事装瓶集团从植物和哺乳动物。组氨酸的抽象可能参与两个non-ionizable质子从底物分子的活性部位。17个已知序列中只有一个组氨酸species-invariant百事装瓶集团的整体序列相似性高。在大肠杆菌百事装瓶集团,这是His128组氨酸。我们在His128执行定点诱变,代之以丙氨酸。突变蛋白H128A催化地活跃。His128组氨酸的一部分——cysteine-rich区域的序列与金属绑定。锌(II)绑定的明显Kd H128A约一个数量级高于野生型蛋白。beplayapp大肠杆菌百事装瓶集团还包含His126是守恒的哺乳动物,真菌和一些细菌的空气层。 We mutated His126 to alanine, and both His126 and His128 simultaneously to alanine. All mutant proteins are catalytically competent; the Vmax values for H128A (44 units/mg), H126A (75 units/mg), and H126/128A (61 units/mg) were similar to wild type PBGS (50 units/mg) in the presence of saturating concentrations of metal ions. The apparent Kd for Zn(II) of H126A and H126/128A is not appreciably different from wild type. The activity of wild type and mutant proteins are all stimulated by an allosteric Mg(II); the mutant proteins all have a reduced affinity for Mg(II). We observe a pKa of approximately 7.5 in the wild type PBGS kcat/Km pH profile as well as in those of H128A and H126/128A, suggesting that this pKa is not the result of protonation/deprotonation of one of these histidines. H128A and H126/128A have a significantly increased Km value for the substrate ALA. This is consistent with a role for one or both of these histidines as a ligand to the required Zn(II) of E. coli PBGS, which is known to participate in substrate binding. Past chemical modification may have inactivated the PBGS by blocking Zn(II) and ALA binding. In addition, the decreased Km for E. coli PBGS at basic pH allows for the quantitation of active sites at four per octamer.