Enzyme-catalyzed acylation of homoserine:: Mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase
Enzyme-catalyzed acylation of homoserine:: Mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase
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DOI：
10.1021/bi000462p
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发表时间：
2000-07-25
期刊：
影响因子：
2.9
通讯作者：
Blanchard, JS
中科院分区：
文献类型：
--
作者：
Born, TL;Franklin, M;Blanchard, JS
细菌和植物甲硫氨酸生物合成的第一个独特步骤涉及高丝氨酸的γ-羟基的酰化。在流感嗜血杆菌中，酰化是通过高丝氨酸转乙酰酶催化的乙酰辅酶A依赖性乙酰化来完成的。该酶的活性调节高丝氨酸进入多种生物合成途径的流量，因此代表细胞生长和活力的关键控制点。我们从 PI 中克隆了高丝氨酸转乙酰酶。流感杆菌并首次对该酶进行了详细的酶学研究。稳态动力学实验表明，该酶利用乒乓动力学机制，其中乙酰辅酶A的乙酰基首先转移至酶亲核体，然后转移至高丝氨酸以形成最终产物O-乙酰高丝氨酸。在测试值范围内，最大速度和 V/K 高丝氨酸与 pH 无关，而 V/K 乙酰辅酶A 取决于 pK 值为 8.6 的单个基团的电离状态，该基团需要质子化。溶剂动力学同位素效应研究对逆反应产生了 0.75 对 V 和 0.74 对 V/K-CoA 的反向影响，对正向反应产生了 1.2 对 V 和 1.7 对 V/K-高丝氨酸的影响。使用快速猝灭标记研究获得了乙酰酶中间体形成的直接证据。基于这些观察，我们提出了酰基转移酶家族这一重要成员的化学机制，并将其机制与高丝氨酸转琥珀酰酶的机制进行了对比。
The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from PI. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K-homoserine were independent of pH over the range of values tested, while V/Kacetyl-CoA was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K-CoA on the reverse reaction and effects of 1.2 on V and 1.7 on V/K-homoserine on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.