Department of Molecular Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
J Biol Chem. 2010 Jan 29;285(5):3351-9. doi: 10.1074/jbc.M109.066548. Epub 2009 Nov 16.
5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 A of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K(m)(SCoA) and k(cat) values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased k(cat) without altering K(m)(SCoA). Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved approximately 3-fold, whereas that of S254T diminished approximately 3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire alpha-oxoamine synthase family of PLP-dependent enzymes.
5-氨基酮戊酸合酶(EC 2.3.1.37)(ALAS)是一种吡哆醛 5'-磷酸(PLP)依赖性酶,在动物、真菌和某些细菌中催化血红素生物合成的初始步骤。甘氨酸和琥珀酰辅酶 A 的缩合产生 5-氨基酮戊酸、辅酶 A 和二氧化碳。Rhodobacter capsulatus ALAS 的 X 射线晶体结构表明,保守的活性位点丝氨酸在封闭的底物结合酶构象中移动到 PLP 辅因子的酚氧原子的氢键距离内,并且在结合的琥珀酰辅酶 A 的硫酯硫原子的 3-4 A 内。为了评估该残基在酶活性中的作用,鼠红细胞 ALAS 中的等效丝氨酸被丙氨酸或苏氨酸取代。尽管 S254A 变体的 K(m)(SCoA)和 k(cat)值分别增加了 25 倍和 2 倍,但 S254T 取代降低了 k(cat)而不改变 K(m)(SCoA)。此外,与野生型 ALAS 相比,S254A 对甘氨酸的催化效率提高了约 3 倍,而 S254T 的催化效率降低了约 3 倍。圆二色性光谱表明,S254A 变体中侧链羟基的去除改变了 PLP 辅因子的微环境并阻碍了琥珀酰辅酶 A 的结合。变体催化反应的瞬态动力学分析和 5-氨基酮戊酸结合时的蛋白质荧光猝灭表明,与产物释放相关的蛋白质构象转变步骤主要受到影响。我们提出以下观点:1)Ser-254 通过将琥珀酰辅酶 A 结合与酶构象平衡偶联,对于形成有能力的催化复合物至关重要,2)活性位点丝氨酸的作用应该扩展到整个依赖 PLP 的α-氧代胺合酶家族的酶。