Elucidation of regulation mechanism of sphingolipid biosynthesis and degradation based on protein structure.

丝氨酸蛋白质转移酶（SPT）催化鞘脂生物合成的第一和速率限制步骤反应。SPT活动的变化直接影响了总数量和细胞鞘磷脂的种类。因此，对SPT的管制机制的采用是非常重要的。（1）我们克隆了鞘磷脂含量细菌的各种SPT基因，并构建了E中SPT的过度表达系统。大肠杆菌，并建立了每个重组SPT的净化方法。（2）我们成功地在一种非反应性模拟的合成中，palmitoyl-CoA，S-（2-oxoheptadecyl）-CoA。使用净化重组SPT，SPT与L-丝氨酸和S-（2-氧戊二基）的反应-CoA在细节中进行了测试。由于L-serin添加到SPT，外部Aldimine中间体通过Michaelis复合体形成。当S-（2-氧戊二基）-CoA添加到这个SPT-L-系列二进制复合体中，它没有转换到最终产品3-酮二氢胰岛素，但 ... More 发现了一个新的Quinonoid中间体吸收峰值。对这一长期复合体的时间解析特性的动力学分析显示了S-（2-氧戊二基）的绑定--CoA对SPT的形成指示。Quinonoid中间体和SPT反应的累积在Quinonoid中间体和原外部aldimine中间体之间的equilibrium。（3）SPT对L-丝氨酸的α-抑制速率，对黄酮类形成速率的相应反应，是核磁共振的检验。展示了L-丝氨酸中Ca的氢-二氢化合物交换在SPT-L-丝氨酸外部铝复合物中缓慢得多，但速率为100-增加了S-（2-氧戊二基）-CoA，显示了SPT中一个显著的物质合成。（4） We succeeded in crystalliization of SPT. SPT·L系列外部铝复合体的结构已被确定。根据晶体学数据，讨论了SPT催化中假定的氨基酸替代方案的作用，这是可能的。Less（低）

Serine palmitoyltransferase (SPT) catalyzes the first and rate-limiting step reaction of the sphingolipid biosynthesis. The change of the SPT activity influences directly the total amount and the kind of the cellar sphingolipids. Therefore, the elucidation of the regulation mechanism of SPT is very important. (1) We cloned the SPT genes of the various kinds of the sphingolipid-content bacteria, constructed the overexpression system of SPT in E. coli, and established the purification method of each recombinant SPT. (2) We succeeded in synthesis of a non-reactive analogue of palmitoyl-CoA, S-(2-oxoheptadecyl)-CoA. Using the purified recombinant SPT, the reaction of SPT with L-serine and S-(2-oxoheptadecyl)-CoA was examined in detail. By the addition of L-serin to SPT, the external aldimine intermediate is formed via the Michaelis complex. When S-(2-oxoheptadecyl)-CoA was added to this SPT-L-serine binary complex, it was not converted to the final product 3-ketodihydrosphingosine, but the … More new absorption peak of the quinonoid intermediate was detected. The kinetic analysis of time resolved spectra of this ternary complex showed that the binding of the S-(2-oxoheptadecyl)-CoA to the SPT induces the formation. And accumulation of the quinonoid intermediate and the SPT reaction is in equilibrium between the quinonid intermediate and the former external aldimine intermediate. (3) The rate of the α-deprotonation of L-serin by SPT, that corresponds to the rate of the quinonoid formation, was examined by NMR. NMR studies showed that the hydrogen-deuterium exchange at Ca of L-serine is very slow in the SPT-L-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. (4) We succeeded in crystallization of SPT. The structure of SPT・L-serine external aldimine complex had been determined. Based on the crystallographic data, the discussion about the role of the conserved amino acid residues in the SPT catalysis became possible. Less