Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.
Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.
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DOI：
10.1016/j.fob.2015.09.009
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发表时间：
2015
期刊：
影响因子：
2.6
通讯作者：
Ferreira GC
中科院分区：
文献类型：
--
作者：
Stojanovski BM;Ferreira GC
琥珀酰辅酶a与ALAS的结合由分子的辅酶a部分促进。阿拉斯的和值有显著差异。在K221V变异中观察到值增加23倍。K221V的增加不是由于琥珀酰辅酶a结合亲和力减弱所致。K221V取代降低了醌类中间产物的生成速率。5-氨基乙酰丙酸合成酶（ALAS）催化哺乳动物血红素生物合成的第一步，即甘氨酸和琥珀酰辅酶a缩合生成辅酶a、CO2和5-氨基乙酰丙酸。荚膜红杆菌（Rhodobacter capsulatus ALAS）的晶体结构表明，琥珀酰辅酶a的腺苷基部分位于一个主要疏水的口袋中，在那里核糖基团与Lys156形成假定的氢键。人类红系阿拉斯（ALAS2）类似赖氨酸的功能丧失突变导致x连锁铁母细胞贫血。为了表征该残基对催化的贡献，我们将小鼠ALAS2中等效的赖氨酸替换为缬氨酸，从而消除了氢键的可能性。K221V的替代产生了23倍的增加和97%的减少。特异性常数的降低并非源于对琥珀酰辅酶a的亲和力较低，因为K221V的亲和力低于野生型ALAS。对于这两种酶，其值与。从底物保护研究进一步推断，K221V对琥珀酰辅酶a具有更强的结合亲和力，在较低琥珀酰辅酶a浓度下，K221V比野生型ALAS具有最大的保护作用。此外，从相同的和值中可以看出，是CoA而不是琥珀基部分促进了琥珀基CoA与野生型ALAS的结合。k221v催化反应的瞬态动力学分析表明，该突变降低了喹诺酮中间体II的生成和衰变速率。总之，结果表明腺苷结合位点Lys221有助于琥珀酰辅酶a的结合和定向，从而有效催化。
Succinyl-CoA binding to ALAS is facilitated by the CoA moiety of the molecule. The and values of ALAS are significantly different. A 23-fold increase in the value was observed with the K221V variant. The increased of K221V is not due to a weakened succinyl-CoA binding affinity. The K221V substitution reduced the rate of quinonoid intermediate formation. 5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linked sideroblastic anemia. To characterize the contribution of this residue toward catalysis, the equivalent lysine in murine ALAS2 was substituted with valine, eliminating the possibility of a hydrogen bond. The K221V substitution produced a 23-fold increase in the and a 97% decrease in . This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the of K221V is lower than that of wild-type ALAS. For both enzymes, the value is significantly different from the . That K221V has stronger binding affinity for succinyl-CoA was further deduced from substrate protection studies, as K221V achieved maximal protection at lower succinyl-CoA concentration than wild-type ALAS. Moreover, it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS, as evident from identical and values. Transient kinetic analyses of the K221V-catalyzed reaction revealed that the mutation reduced the rates of quinonoid intermediate II formation and decay. Altogether, the results imply that the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis.