Yang S Y, Bittman R, Schulz H
J Biol Chem. 1985 Mar 10;260(5):2862-8.
The kinetic properties of the fatty acid oxidation complex from Escherichia coli were studied with the aim of elucidating the functional consequence of having enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase associated with a multifunctional polypeptide. The kinetic parameters of individual enzymes were determined and used in model calculations based on a published theory (Storer, A. C., and Cornish-Bowden, A. (1974) Biochem. J. 141, 205-209) to predict the kinetic behavior of a system of functionally unlinked enzymes. The validity of the theory for making these calculations was proven by demonstrating a good agreement between the calculated and observed rates of intermediate and product formation for the conversion of 2-decenoyl-CoA to 3-ketodecanoyl-CoA catalyzed by a mixture of bovine liver enoyl-CoA hydratase and pig heart L-3-hydroxyacyl-CoA dehydrogenase. The conversion of 2-decenoyl-CoA to 3-ketodecanoyl-CoA catalyzed by the sequential action of the hydratase and dehydrogenase of the complex from E. coli was determined by measuring the rate of NADH formation. Stopped-flow measurements showed the rate of NADH formation to be linear without any lag period. When the initial velocity of the hydratase was 10.2 microM min-1, that of the overall reaction was 8.41 microM min-1. In contrast, the results calculated by use of the Storer and Cornish-Bowden equation for a system of unlinked enzymes predicted the overall reaction to exhibit a lag time of 30 s and to result in the accumulation of 2.1 microM 3-hydroxydecanoyl-CoA before reaching a velocity corresponding to 82.5% of that of the hydratase reaction. The high initial rate and the unusual kinetic properties of the overall reaction observed in the present study are best explained by a channeling mechanism on the large subunit of the E. coli fatty acid oxidation complex. When the apparent degree of channeling is corrected for the percentage of the dehydrogenase active sites saturated with NAD+, more than 90% of the intermediate appears to be transferred directly from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase.
对来自大肠杆菌的脂肪酸氧化复合物的动力学性质进行了研究，目的是阐明烯酰辅酶A水合酶和3-羟酰基辅酶A脱氢酶与多功能多肽结合的功能后果。测定了各个酶的动力学参数，并将其用于基于已发表理论（斯托勒，A.C.，和康沃尔-鲍登，A.（1974年）《生物化学杂志》141，205 - 209）的模型计算，以预测功能未连接的酶系统的动力学行为。通过证明在牛肝烯酰辅酶A水合酶和猪心L-3-羟酰基辅酶A脱氢酶混合物催化2-癸烯酰辅酶A转化为3-酮癸酰辅酶A的过程中，计算得到的中间产物和产物形成速率与观察到的速率之间有良好的一致性，证明了该理论用于这些计算的有效性。通过测量NADH形成速率，确定了大肠杆菌复合物中的水合酶和脱氢酶顺序作用催化2-癸烯酰辅酶A转化为3-酮癸酰辅酶A的过程。停流测量表明NADH形成速率呈线性，没有任何延迟期。当水合酶的初始速度为10.2微摩尔/分钟时，总反应的初始速度为8.41微摩尔/分钟。相比之下，使用斯托勒和康沃尔-鲍登方程对未连接的酶系统进行计算的结果预测，总反应将表现出30秒的延迟期，并在达到相当于水合酶反应速度82.5%的速度之前积累2.1微摩尔的3-羟癸酰辅酶A。本研究中观察到的总反应的高初始速率和不寻常的动力学性质，最好用大肠杆菌脂肪酸氧化复合物大亚基上的通道化机制来解释。当对通道化的表观程度进行校正，以考虑脱氢酶活性位点被NAD +饱和的百分比时，超过90%的中间产物似乎直接从烯酰辅酶A水合酶的活性位点转移到3-羟酰基辅酶A脱氢酶的活性位点。
