Allard J, Grochulski P, Sygusch J
Département de Biochimie, Université de Montréal, Montreal, QC, H3C 3J7 Canada.
Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3679-84. doi: 10.1073/pnas.071380898.
2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-A resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an epsilon-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.
2-酮-3-脱氧-6-磷酸葡萄糖酸（KDPG）醛缩酶催化KDPG可逆裂解为丙酮酸和3-磷酸甘油醛。该酶属于I类醛缩酶，其反应机制涉及Lys-133与酮底物之间席夫碱中间体的形成。在pH 4.6的酸性条件下，用10 mM丙酮酸浸泡KDPG醛缩酶晶体，通过快速冷冻捕获了一种共价加合物。分辨率为1.95 Å的结构测定表明，丙酮酸与Lys-133发生了亲核攻击，形成了质子化的氨基醇中间体，这是一种功能性席夫碱前体，通过与活性位点残基的氢键作用得以稳定。氨基醇与Glu-45的相互作用表明了几个速率步骤的一般碱催化作用。Phe-135与丙酮酸甲基的芳香相互作用确保了立体特异性加成。在天然结构中，Lys-133提供了其所有的氢键，表明存在一个ε-铵盐基团。推测亲核活化是通过单质子化两性离子对（Glu-45/Lys-133）中的质子转移发生的。两性离子对的形成需要质子化的Lys-133预先进行侧链重排，以取代与两性离子残基氢键结合的水分子。
