Haas J A, Fox B G
The Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison, Wisconsin 53705, USA.
Biochemistry. 1999 Sep 28;38(39):12833-40. doi: 10.1021/bi991318a.
Stearoyl acyl carrier protein Delta(9) desaturase (Delta9D) uses a diiron center to catalyze the NADPH- and O(2)-dependent desaturation of stearoyl acyl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ricinus communis Delta9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the specificity for position of double-bond insertion, the kinetic parameters for the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O(2) consumption and olefin production was found to be maximal for 18:0-ACP, and the loss of coupling observed for the more slowly desaturated acyl-ACPs was attributed to autoxidation of the electron-transfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the K(M) values were similar ( approximately 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by approximately 26-fold for the same range of acyl chain lengths. A linear increase in log (k(cat)/K(M)) was observed upon lengthening of the acyl chain from 15:0- to 18:0-ACP, while no further increase was observed for 19:0-ACP. The similarity of the k(cat)/K(M) values for 18:0- and 19:0-ACPs and the retained preference for double-bond insertion at the Delta(9) position with 19:0-ACP (>98% desaturation at the Delta(9) position) suggest that the active-site channel past the diiron center can accommodate at least one more methylene group than is found in the natural substrate. The DeltaDeltaG(binding) estimated from the change in k(cat)/K(M) for increasing substrate acyl-chain length was -3 kJ/mol per methylene group, similar to the value of -3.5 kJ/mol estimated for the hydrophobic partition of long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R. , and Tanford, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 289-293]. Since the K(M) values are overall similar for all acyl-ACPs tested, the progressive increase in hydrophobic binding energy available from increased chain length is apparently utilized to enhance catalytic steps, which thus provides the underlying physical mechanism for acyl chain selectivity observed with Delta9D.
硬脂酰酰基载体蛋白Δ9去饱和酶（Δ9D）利用双铁中心催化硬脂酰酰基载体蛋白（ACP）依赖于NADPH和O₂的去饱和反应，生成油酰-ACP。利用重组蓖麻Δ9D与天然和非天然链长的酰基-ACP的反应，研究了重组酶复合物的偶联、双键插入位置的特异性、去饱和反应的动力学参数以及对酰基链长度的选择性。发现NADPH和O₂消耗与烯烃生成的偶联在18:0-ACP时最大，而对于去饱和较慢的酰基-ACP观察到的偶联损失归因于电子传递链的自氧化。对具有不同酰基链长度的酰基-ACP去饱和的稳态动力学参数分析表明，对于15:0-18:0-ACP，K(M)值相似（相差约2.5倍），而在相同酰基链长度范围内，k(cat)值增加了约26倍。当酰基链从15:0延长到18:0-ACP时，log(k(cat)/K(M))呈线性增加，而对于19:0-ACP则未观察到进一步增加。18:0-和19:0-ACP的k(cat)/K(M)值相似，且19:0-ACP在Δ9位置双键插入的偏好性得以保留（在Δ9位置去饱和>98%），这表明经过双铁中心的活性位点通道能够容纳比天然底物中多至少一个亚甲基。根据增加底物酰基链长度时k(cat)/K(M)的变化估算的ΔΔG(binding)为每亚甲基-3 kJ/mol，类似于从水到庚烷的长链脂肪酸（C-7至C-21）疏水分配估算的-3.5 kJ/mol的值[史密斯，R.，和坦福德，C.（1973年）美国国家科学院院刊70, 289 - 293]。由于对于所有测试的酰基-ACP，K(M)值总体相似，增加链长度可获得的疏水结合能的逐渐增加显然被用于增强催化步骤，这从而为Δ9D观察到的酰基链选择性提供了潜在的物理机制。
