Role of arginine 439 in substrate binding of 5-aminolevulinate synthase.
Role of arginine 439 in substrate binding of 5-aminolevulinate synthase.
复制标题
精氨酸 439 在 5-氨基乙酰丙酸合酶底物结合中的作用。
DOI：
10.1021/bi971928f
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Ferreira,GC
中科院分区：
文献类型：
--
作者：
Tan,D;Harrison,T;Hunter,GA;Ferreira,GC
5-氨基乙酰丙酸合成酶（Aminolevulinate synthase，EC 2.3.1.37）是非植物真核生物和一些原核生物血红素生物合成途径中的第一步反应。5-氨基乙酰丙酸合成酶与其他α-家族吡哆醛5 '-磷酸依赖性酶的同源性序列建模表明，小鼠红系5-氨基乙酰丙酸合成酶的Arg-439残基是该家族吡哆醛5'-磷酸依赖性酶中的保守残基。此外，这种保守的精氨酸残基在几种酶中，天冬氨酸氨基转移酶（其三维结构是已知的）已显示与底物羧基相互作用。为了测试Arg-439是否参与鼠红系5-氨基乙酰丙酸合酶中的底物结合，使用定点诱变将鼠红系5-氨基乙酰丙酸合酶的Arg-439和Arg-433各自替换为Lys和Leu。R439 K突变体保留了野生型活性的77%;它对两种底物的Km值增加了9 - 13倍，而R433 K的活性增加了2倍，两种底物的Km值保持不变。如使用标准5-氨基乙酰丙酸合酶酶偶联活性测定所测定，R439 L没有可测量的活性。相反，R433 L的动力学参数与野生型的动力学参数相当。R439 K和R439 L突变体对甘氨酸的解离常数（Kd）分别增加了5倍和30倍以上，而R433 K和R433 L突变体对甘氨酸的Kd值与野生型相似。然而，除了R439 L的Kd增加10倍外，突变体和野生型之间的甲胺结合没有太大差异。R439 K的热稳定性比野生型酶低得多，热转变温度T1/2比野生型酶低8.3 °C。此外，体内互补分析表明，在鼠红系5-氨基乙酰丙酸合酶的活性位点中，R439由与K313（其参与吡哆醛5 '-磷酸辅因子的席夫碱键）和D279（其与辅因子的环氮静电相互作用）相同的亚基贡献，而另一个亚基提供R149。总而言之，这些发现表明Arg-439在小鼠红细胞5-氨基酮戊酸合成酶的底物结合中发挥重要作用。
5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other α-family pyridoxal 5‘-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5‘-phosphate-dependent enzymes. Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group. To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis. The R439K mutant retained 77% of the wild-type activity; itsKmvalues for both substrates increased 9−13-fold, while the activity of R433K increased 2-fold and theKmvalues for both substrates remained unchanged. R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay. In contrast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, whileKdvalues for glycine for both R433K and R433L mutants were similar to those of the wild-type. However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase inKdmethylaminefor R439L. R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature,T1/2, determined to be 8.3 °C lower than that of the wild-type enzyme. In addition,in vivocomplementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5‘-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149. Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.