Probing the catalytic mechanism of yeast triose phosphate isomerase by site-specific mutagenesis.
Probing the catalytic mechanism of yeast triose phosphate isomerase by site-specific mutagenesis.
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通过定点诱变探讨酵母磷酸丙糖异构酶的催化机制。
DOI：
10.1042/bst0120229
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发表时间：
1984
影响因子：
3.9
通讯作者：
RaiBhandary,UL
中科院分区：
文献类型：
--
作者：
Petsko,GA;DavenportJr,RC;Frankel,D;RaiBhandary,UL
酵母糖酵解酶磷酸丙糖异构酶催化一种非常简单的反应，即一个质子的运动以相互转化 DHAP 和 GAP。该酶是两个相同亚基的二聚体，每个亚基均为 M, 27000，并且没有辅因子或所需的金属离子。子单元之间不存在协同作用。在哈佛大学医学院 Dan Fraenkel 教授的协助下，酿酒酵母磷酸丙糖异构酶基因已被克隆并测序（Alber & Kawasaki, 1982）。使用衍生的蛋白质序列，我们还通过 0.19 nm 分辨率的 X 射线衍射确定了酶的完整三维结构（Alber 等人，1983b；T. Alber、M. Rose 和 G. A. Petsko，未发表的作品），并通过限制最小二乘法将结构细化至 R 因子为 0.31。在晶体学家看来，磷酸丙糖异构酶是一种完美的催化剂，原因如下：与水解酶的产物从酶中快速释放并且热力学上不利于反向反应不同，这里正向和反向反应都非常快，并且平衡以20比1的比例有利于DHAP的形成；当结晶异构酶被给予过量的底物时，可以对酶结合的物质进行晶体学研究。对于酵母酶，当 DHAP 在 10°C 下扩散到晶体中时，米氏复合体的可视化分辨率为 0.35 nm（Alber 等人，1981；T. Alber、G. A. Petsko & M. Rose，未发表的作品）。目前正在进行的工作是研究与过渡态类似物磷酸乙二醇异羟肟酸共结晶的酶的不同晶型；这些晶体的衍射分辨率为 0.16 nm（T. Alber、D. Ringe Ponzi 和 G. A. Petsko，未发表的作品）。我们和其他人根据我们目前的结构知识提出了该反应的机制。晶体学和化学证据表明，Glu-165 的羧酸侧链是在底物的 C-1 和 C-2 之间转移质子的催化碱基；考虑到这一点，化学问题就是稳定碳上正在形成的负电荷。该电荷通过共振共享到底物的氧原子中，氧原子位于活性位点的亲电子基团旁边。
The yeast glycolytic enzyme triose phosphate isomerase catalyses a very simple reaction, the movement of one proton to interconvert DHAP and GAP. The enzyme is a dimer of two identical subunits each of M, 27000, and has no cofactors or required metal ions. There is no co-operativity between the subunits. With the assistance of Professor Dan Fraenkel of the Harvard University Medical School, the gene for Saccharomyces cereuisiae triose phosphate isomerase has been cloned and sequenced (Alber & Kawasaki, 1982). Using the derived protein sequence we have also determined the complete three-dimensional structure of the enzyme by X-ray diffraction at 0.19 nm resolution (Alber et al., 1983b; T. Alber, M. Rose & G. A. Petsko, unpublished work), and have refined the structure by restrained-least-squares methods to an R-factor of 0.31. In the view of the crystallographer, triose phosphate isomerase is a perfect catalyst for the following reason: unlike a hydrolytic enzyme where the products are rapidly released from the enzyme and the back reaction is thermodynamically disfavoured, here the forward and back reactions are both very rapid, and the equilibrium favours the formation of DHAP by 20 to 1; when crystalline isomerase is given excess substrate, the enzyme-bound species can be studied crystallographically. In the case of the yeast enzyme, the Michaelis complex was visualized to 0.35 nm resolution when DHAP was diffused into the crystal at-10 C (Alber et al., 1981; T. Alber, G. A. Petsko & M. Rose, unpublished work). Current work is underway to study a different crystal form of the enzyme co-crystallized with the transition state analogue phosphoglycolohydroxamic acid; these crystals diffract to 0.16 nm resolution (T. Alber, D. Ringe Ponzi & G. A. Petsko, unpublished work). We and others have suggested a mechanism for the reaction on the basis of our current structural knowledge.Crystallographic and chemical evidence suggest that the carboxylate side chain of Glu-165 is the catalytic base which transfers the proton between C-1 and C-2 of the substrate; with this in mind, the chemical problem is then to stabilize the developing negative charge on the carbon. This charge is shared through resonance into the oxygen atoms of the substrate, which are positioned next to electrophilic groups in the active site.