Davenport R C, Bash P A, Seaton B A, Karplus M, Petsko G A, Ringe D
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1991 Jun 18;30(24):5821-6. doi: 10.1021/bi00238a002.
The glycolytic enzyme triosephosphate isomerase (TIM) catalyzes the interconversion of the three-carbon sugars dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) at a rate limited by the diffusion of substrate to the enzyme. We have solved the three-dimensional structure of TIM complexed with a reactive intermediate analogue, phosphoglycolohydroxamate (PGH), at 1.9-A resolution and have refined the structure to an R-factor of 18%. Analysis of the refined structure reveals the geometry of the active-site residues and the interactions they make with the inhibitor and, by analogy, the substrates. The structure is consistent with an acid-base mechanism in which the carboxylate of Glu-165 abstracts a proton from carbon while His-95 donates a proton to oxygen to form an enediol (or enediolate) intermediate. The conformation of the bound substrate stereoelectronically favors proton transfer from substrate carbon to the syn orbital of Glu-165. The crystal structure suggests that His-95 is neutral rather than cationic in the ground state and therefore would have to function as an imidazole acid instead of the usual imidazolium. Lys-12 is oriented so as to polarize the substrate oxygens by hydrogen bonding and/or electrostatic interaction, providing stabilization for the charged transition state. Asn-10 may play a similar role.
糖酵解酶磷酸丙糖异构酶（TIM）催化二羟丙酮磷酸（DHAP）和D-甘油醛3-磷酸（GAP）这两种三碳糖之间的相互转化，其反应速率受底物向酶扩散的限制。我们已解析出与反应性中间类似物磷酸甘油异羟肟酸（PGH）复合的TIM的三维结构，分辨率为1.9埃，并将该结构精修至R因子为18%。对精修结构的分析揭示了活性位点残基的几何结构以及它们与抑制剂的相互作用，由此类推，也揭示了与底物的相互作用。该结构与酸碱机制一致，即Glu-165的羧酸盐从碳原子上夺取一个质子，而His-95向氧原子提供一个质子以形成烯二醇（或烯二醇盐）中间体。结合底物的构象在立体电子学上有利于质子从底物碳原子转移至Glu-165的顺式轨道。晶体结构表明，His-95在基态时呈中性而非阳离子状态，因此必须作为咪唑酸而非通常的咪唑鎓发挥作用。Lys-12的取向使其通过氢键和/或静电相互作用使底物氧原子极化，为带电荷的过渡态提供稳定性。Asn-10可能发挥类似作用。
