Choi K H, Shi J, Hopkins C E, Tolan D R, Allen K N
Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215, USA.
Biochemistry. 2001 Nov 20;40(46):13868-75. doi: 10.1021/bi0114877.
Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.
果糖-1,6-双（磷酸）醛缩酶是一种在所有脊椎动物和高等植物中都存在的必需糖酵解酶，它催化果糖1,6-双（磷酸）（Fru-1,6-P₂）裂解为3-磷酸甘油醛和磷酸二羟丙酮（DHAP）。人类醛缩酶基因的突变会导致溶血性贫血和遗传性果糖不耐受。通过在Fru-1,6-P₂存在下用NaBH₄捕获催化中间体，已通过X射线晶体学将醛缩酶-DHAP席夫碱的结构解析到2.6 Å分辨率（R₍cryst₎ = 0.213，R₍free₎ = 0.249）。这是这种必需糖酵解酶捕获的共价中间体的首个结构。该结构有助于阐明全面的催化机制，并识别席夫碱醛缩酶中保守的化学基序。结合的DHAP相对于Asp33的位置与Asp33在使C4-羟基去质子化从而导致C-C键裂解中所起的作用一致。Ala31的甲基侧链直接位于C3-羟基的对面，在空间上有利于该碳原子上底物的S-构型。结合底物C6-磷酸基团的“触发”残基Arg303是DHAP磷酸基团的配体。观察到的配体在底物和产物磷酸基团之间的移动可能为底物裂解与C末端与产物释放相关的构象变化之间提供结构联系。Glu187相对于DHAP席夫碱的位置与该残基在脱水步骤中催化席夫碱形成时对甲醇胺羟基进行质子化所起的作用一致。将醛缩酶-DHAP结构与机制相似的转醛醇酶和KDPG醛缩酶的共价酶-二羟丙酮结构进行叠加，可识别参与席夫碱形成和分解的保守赖氨酸-谷氨酸二元组。叠加突出了这样一个事实，即醛缩酶中的Lys146在转醛醇酶中被Asn35取代。转醛醇酶中的这种取代稳定了第二个醛糖底物攻击所需的烯胺中间体，将化学性质从醛缩酶转变为转醛醇酶。
