Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase.
Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase.
复制标题
硬脂酰-ACP 去饱和酶中酶-底物复合物形成的荧光各向异性研究。
DOI：
10.1021/bi020340s
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Fox,BrianG
中科院分区：
文献类型：
--
作者：
Haas,JeffreyA;Fox,BrianG
硬脂酰酰基载体蛋白 Δ9-去饱和酶 (Δ9D) 催化顺式双键区域和立体特异性插入到附着于酰基载体蛋白的酰基链中。使用酰化形式的丹酰基和荧光素基 ACP 进行稳态和停流荧光各向异性测量，揭示了 16:0-、17:0- 和 18:0-ACP 的平衡解离常数和解离速率常数，其中 Δ9D 处于静止状态和化学 4e 还原状态。观察到 1 nM 18:0-荧光素基-ACP 与二聚体静止 Δ9D 的一个亚基的结合，KD1= 13 ± 3 nM。 4e-Δ9D 的 KD1 值没有观察到显着差异。将酰基链从 18:0 缩短至 17:0，然后缩短至 16:0 时，观察到每个亚甲基的 KD1 增加了约 4 倍。在使用 850 nM 18:0-dansyl-ACP 进行的不同实验中，与静息 Δ9D 的第二个亚基的结合估计具有 KD2≈ 350 ± 40 nM。 KD2 值表现出与 KD1 值观察到的类似的酰基链长度依赖性。通过停流各向异性测量来测量酶-底物复合物逆转的 koff 值也依赖于酰基链长度，并且相对于 18:0-ACP (1 s-1)，16:0-ACP (130 s-1) 增加了 130 倍。因此，酰基链长度的增加与目前报道的KD和koff值的增加相关。这些结果表明，酰基链长度选择性主要源自酶-底物复合物在底物释放和后续催化步骤之间的分配。
Stearoyl-acyl carrier protein Δ9-desaturase (Δ9D) catalyzes regio- and stereospecific insertion ofcisdouble bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e-reduced Δ9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting Δ9D was observed withKD1= 13 ± 3 nM. No significant difference in theKD1value was observed for 4e-Δ9D. An ∼4-fold increase inKD1per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting Δ9D was estimated to haveKD2≈ 350 ± 40 nM. TheKD2values exhibited a similar dependence on acyl chain length as observed for theKD1values. Thekoffvalues measured by stopped-flow anisotropy measurements for reversal of the enzyme−substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s-1) relative to 18:0-ACP (1 s-1). Increases in acyl chain length are thus associated with the presently reported increases in theKDandkoffvalues. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme−substrate complex between substrate release and subsequent steps in catalysis.