Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors
Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors
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DOI：
10.1021/bi035706v
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发表时间：
2004-02-03
期刊：
影响因子：
2.9
通讯作者：
Kagamiyama, H
中科院分区：
文献类型：
--
作者：
Ikushiro, H;Hayashi, H;Kagamiyama, H
丝氨酸棕榈酰转移酶（Serine palmitoyltransferase，SPT）是鞘脂生物合成的关键酶，催化L-丝氨酸和棕榈酰辅酶A脱羧缩合生成3-酮二氢鞘氨醇。我们成功地在大肠杆菌中从少动鞘氨醇单胞菌EY 2395（T）中过量生产了一种水溶性同源二聚体SPT。重组SPT显示了其辅酶吡哆醛5 '-磷酸的特征吸收和圆二色谱。在此基础上，我们分析了SPT与L-丝氨酸及其产物的反应，发现了以下新的方面：第一，我们分析了L-丝氨酸与3-羟基丙酸的结合，发现底物引起SPT的光谱变化是由于形成了一个外部的醛亚胺中间体，而不是米氏络合物。其次，还检查了各种丝氨酸类似物;数据表明，L-丝氨酸的α-羧基对于SPT的底物识别非常重要。第三，我们重点介绍了一系列SPT抑制剂，这些抑制剂已被用作研究鞘脂耗竭引起的细胞反应的方便工具。SPT与多球壳菌素的相互作用表明，这种产品相关的化合物会强烈地和竞争性地抑制酶的活性，通过在酶的活性位点形成外部醛亚胺。发现β-氯-L-丙氨酸和L-环丝氨酸产生特征性的PLP-加合物，其以不可逆的方式产生SPT的失活。对SPT失活的详细机制进行了讨论。这是首次分析这些化合物对SPT的抑制机制，这将为解释细胞生物学实验的结果提供酶学基础。
Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation Of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395(T) in Escherichia coli. The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5'-phosphate. On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to L-serine and product, and showed the following new aspects: First, we analyzed the binding of L-serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex. Second, various serine analogues were also examined; the data indicated that the alpha-carboxyl group of L-serine was quite important for substrate recognition by SPT. Third, we focused on a series of SPT inhibitors, which have been used as convenient tools to study the cell responses caused by sphingolipid depletion. The interaction of SPT with myriocin suggested that such product-related compounds would strongly and competitively inhibit enzyme activity by forming an external aldimine in the active site of the enzyme. beta-Chloro-L-alanine and L-cycloserine were found to generate characteristic PLP-adducts that produced inactivation of SPT in an irreversible manner. The detailed mechanisms for the SPT inactivation were discussed. This is the first analysis of the inhibition mechanisms of SPT by these compounds, which will provide an enzymological basis for the interpretation of the results from cell biological experiments.