Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase*
Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase*
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His159 在丝氨酸棕榈酰转移酶催化反应中的多功能作用*
DOI：
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发表时间：
2009
影响因子：
4.8
通讯作者：
H. Hayashi
中科院分区：
文献类型：
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作者：
Y. Shiraiwa;H. Ikushiro;H. Hayashi
丝氨酸棕榈酰基转移酶(SPT)属于5‘-磷酸吡哆醛(PLP)依赖酶的I型折叠家族，由L-丝氨酸和棕榈酰辅酶A合成3-酮二氢鞘氨醇(KDS)。与其他α-草胺合成酶亚家族酶一样，Spt与大多数折叠I型酶的不同之处在于其表面由His残基(His159)占据，而不是芳香氨基酸残基。将His159转变为丙氨酸或芳香族氨基酸残基，以考察其在催化过程中的作用。突变型SPT均能形成解离常数为野生型的10倍以上的PLP-L-丝氨酸乙二胺，并催化L-丝氨酸转氨基的流产。这些结果表明，His159不仅是L-丝氨酸的锚定位点，而且通过固定PLP-L-丝氨酸乙二胺的构象来调节L-丝氨酸的α去质子化，以防止不必要的副反应。只有H159A SPT在催化过程中保持了活性，并显示出明显的喹酮类化合物的505 nm吸收带。时间分辨光谱的全局分析表明，存在两个醌中间体，第一个来自PLP-L-丝氨酸乙二胺，第二个来自PLP-KDS乙二胺。这些苯醌中间体的积累表明，His159既促进了作为酸催化剂的克莱森型缩合反应，也促进了第二醌类化合物在Cα处的质子化生成PLP-KDS醛二胺。这些结果，结合先前的模型构建研究(Ikushiro，H.，Fujii，S.，Shiraiwa，Y.，和Hayashi，H.(2008)J.Biol)。化学。283,7542-7553)，导致我们提出了一种新的机制，其中His159通过利用Dunathan猜想的立体化学扮演了多重角色。
Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His159 is not only the anchoring site for l-serine but regulates the α-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His159 promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of Dunathan's conjecture.
通过用丙氨酸替换 Asp222 来表征大肠杆菌天冬氨酸转氨酶中诱导的表观负协同性。
DOI：
10.1093/protein/7.3.413
发表时间：
1994
期刊：
Protein engineering
影响因子：
--
作者：
Onuffer,JJ;Kirsch,JF
通讯作者：
Kirsch,JF