Desaturation, chain scission, and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase.
Desaturation, chain scission, and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase.
复制标题
DOI：
10.1021/bi020306d
复制
发表时间：
2002-08
期刊：
影响因子：
2.9
通讯作者：
C. Rogge;B. Fox
中科院分区：
文献类型：
--
作者：
C. Rogge;B. Fox
硬脂酰酰基载体蛋白Delta（9）去饱和酶催化在动力学上优选的天然底物18：0-ACP中酰基链的C-9和C-10位置之间的顺式双键的NADPH和O（2）依赖性插入。在这项工作中，底物类似物与氧原子单独取代亚甲基基团在8，9，10，和11位的硬脂酰链合成，转化为酰氧基-ACP，并用作探针的去饱和酶的反应性。去饱和，酰氧基断链，并在结合之前，断链寄存器移位的证据。与具有O-8或O-11取代基的酰氧基-ACP的反应得到与在C-9和C-10之间插入顺式双键一致的单一去饱和产物。O-8-和O-11-取代的酰氧基-ACP的k（cat）/K（M）值与天然底物的值相当，表明反应位点附近醚基的存在不会显著干扰去饱和反应或C-9和C-10之间去饱和的适当寄存器中底物的结合。对于与O-9和O-10酰氧基-ACP的反应，k（cat）值降低至18：0-ACP所观察到的值的约3%，并且在反应时，酰氧基链断裂以产生ω-羟基脂肪烷酰基-ACP和挥发性长链醛。对于O-9取代，获得8-羟基辛酸酯和1-壬醛，对应于预期的结合寄存器和O-9和C-10位置之间的后续反应。相比之下，O-10取代产生9-羟基壬酰基-ACP和1-辛醛，对应于O-10和C-11位置之间反应之前酰氧基链结合的专性“寄存器移位”。因此，寄存器移位被定义为酰基链结合的机械相关的未对准，其导致在C-9和C-10之间以外的位置处的反应。O-10酰氧基探针不能在C-9和O-10位置之间进行反应，这提供了证据，证明硬脂酰-ACP的Δ 9D催化的去饱和作用可能在C-10开始。被认为是可能的酰基断链的机制和这些结果的去饱和机制的影响。
Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.