The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.
The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.
复制标题
酪氨酸 121 在 5-氨基乙酰丙酸合酶的辅因子结合中的作用。
DOI：
10.1002/pro.5560070516
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;Barber,MJ;Ferreira,GC
5‐Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5′‐phosphate as an essential cofactor. Tyr‐121 is a conserved residue in all known sequences of 5‐aminolevulinate synthases. Further, it corresponds to Tyr‐70 ofEscherichia coliaspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr‐121 is involved in cofactor binding in murine erythroid 5‐aminolevulinate synthase, Tyr‐121 of murine erythroid 5‐aminolevulinate synthase was substituted by Phe and His using site‐directed mutagenesis. The Y121F mutant retained 36% of the wild‐type activity and theKmvalue for substrate glycine increased 34‐fold, while the activity of the Y121H mutant decreased to 5% of the wild‐type activity and theKmvalue for glycine increased fivefold. The pKalvalues in the pH‐activity profiles of the wild‐type and mutant enzymes were 6.41, 6.54, and 6.65 for wild‐type, Y121F, and Y121H, respectively. The UV‐visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild‐type with the exception of an absorption maximum shift (420 ∼ 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild‐type for the cofactor, reflected in theKdvalues for pyridoxal 5′‐phosphate (26.5, 6.75, and 1.78 μ. for Y121F, Y121H, and the wild‐type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr‐121 plays a critical role in cofactor binding of murine erythroid 5‐aminolevulinate synthase.
