Kinetic investigations of the rate-limiting step in human 12-and 15-lipoxygenase
Kinetic investigations of the rate-limiting step in human 12-and 15-lipoxygenase
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DOI：
10.1021/bi0273462
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发表时间：
2003-05-13
期刊：
影响因子：
2.9
通讯作者：
Holman, TR
中科院分区：
文献类型：
--
作者：
Segraves, EN;Holman, TR
哺乳动物脂肪氧合酶与几种炎症性疾病有关，然而，其动力学机制的细节仍不清楚。本文分别用花生四烯酸（AA）和亚油酸（LA）对人血小板12-脂氧合酶（12-hLO）和人网织红细胞15-脂氧合酶-1（15-hLO）进行了测定，并在温度、溶解氧浓度和粘度等实验条件下进行了比较。所呈现的数据显示，12-hLO和15-hLO具有比大豆脂氧合酶-1（sLO-1）更慢的产物释放速率（k（cat）），但是对于脂肪酸（k（cat）/K-M）或分子氧[k（cat）/K-M（O-2）]具有相似或更好的底物捕获速率。在10-35 ℃范围内，15-hLO与LA的主要动力学同位素效应（KIE）被确定为与温度无关且很大（（D）k（cat）= 40 +/- 8），表明C-H键断裂是唯一的限速步骤，并通过隧道机制进行。然而，15-hLO的（D）k（cat）/K-M是温度依赖性的，与我们以前的结果一致[刘易斯，E. R.，Johansen，E.，Holman，T. R.等人（1999）J. Am. 121，1395-1396]，表明多个限速步骤。这通过温度依赖性的k（cat）/K-M溶剂同位素效应（SIE）证实，其表明在低温下的氢键重排步骤，类似于sLO-1的步骤[Glickman，M. H、和Klinman，J.P.（1995）Biochemistry 34，14077-14092]。由于12-hLO不能有效催化LA，因此无法测定其KIE，但k（cat）/K-M SIE与温度无关，表明15-hLO和sLO-1均存在不同的速率限制步骤。
Mammalian lipoxygenases have been implicated in several inflammatory disorders; however, the details of the kinetic mechanism are still not well understood. In this paper, human platelet 12-lipoxygenase (12-hLO) and human reticulocyte 15-lipoxygenase-1 (15-hLO) were tested with arachidonic acid (AA) and linoleic acid (LA), respectively, under a variety of changing experimental conditions, such as temperature, dissolved oxygen concentration, and viscosity. The data that are presented show that 12-hLO and 15-hLO have slower rates of product release (k(cat)) than soybean lipoxygenase-1 (sLO-1), but similar or better rates of substrate capture for the fatty acid (k(cat)/K-M) or molecular oxygen [k(cat)/K-M(O-2)]. The primary, kinetic isotope effect (KIE) for 15-hLO with LA was determined to be temperature-independent and large ((D)k(cat) = 40 +/- 8), over the range of 10-35 degreesC, indicating that C-H bond cleavage is the sole rate-limiting step and proceeds through a tunneling mechanism. The (D)k(cat)/K-M for 15-hLO, however, was temperature-dependent, consistent with our previous results [Lewis, E. R., Johansen, E., and Holman, T. R. (1999) J. Am. Chem. Soc. 121, 1395-1396], indicating multiple rate-limiting steps. This was confirmed by a temperature-dependent, k(cat)/K-M solvent isotope effect (SIE), which indicated a hydrogen bond rearrangement step at low temperatures, similar to that of sLO-1 [Glickman, M. H., and Klinman, J. P. (1995) Biochemistry 34, 14077-14092]. The KIE could not be determined for 12-hLO due to its inability to efficiently catalyze LA, but the k(cat)/K-M SIE was temperature-independent, indicating distinct rate-linuting steps from both 15-hLO and sLO-1.