El Bawab S, Birbes H, Roddy P, Szulc Z M, Bielawska A, Hannun Y A
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, South Carolina 29425, USA.
J Biol Chem. 2001 May 18;276(20):16758-66. doi: 10.1074/jbc.M009331200. Epub 2001 Feb 8.
We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.
我们之前已从大鼠脑中纯化出一种膜结合神经酰胺酶，最近克隆了其人类同源物。我们还观察到，同一酶能够催化神经酰胺合成的逆反应。为深入了解该酶的生物化学性质，我们在本研究中对这种逆活性进行了表征。以鞘氨醇和棕榈酸为底物时，该酶呈现米氏动力学；然而，该酶不利用棕榈酰辅酶A作为底物。此外，伏马菌素B1（一种依赖辅酶A的神经酰胺合酶抑制剂）在体外和细胞内均未抑制该活性。该酶在中性范围内表现出较窄的最适pH值，在碱性范围内活性非常低。进行了底物特异性研究，该酶对d-赤藓型鞘氨醇表现出最高活性（Km为0.16 mol%，Vmax为0.3微摩尔/分钟/毫克），但d-赤藓型二氢鞘氨醇和鞘氨醇的三种非天然立体异构体是较差的底物。还研究了对脂肪酸的特异性，观察到肉豆蔻酸的活性最高，Km为1.7 mol%，Vmax为0.63微摩尔/分钟/毫克。进行动力学研究以探究反应机制，林-贝氏图表明是顺序机制。鉴定出两种底物的两种竞争性抑制剂，l-赤藓型鞘氨醇和肉豆蔻醛，抑制研究表明该反应遵循随机顺序机制。还测试了脂质效应。这些脂质大多表现出中度抑制，而磷脂酸和心磷脂的作用更强，在约2.5 - 5 mol%时完全抑制。矛盾的是，心磷脂刺激了神经酰胺酶活性。这些结果确定了这种逆活性的生物化学特征。鉴于该酶可能受细胞内pH值调节或与心磷脂和/或磷脂酸相互作用，对结果进行了讨论。
