Choi Kyung H, Tolan Dean R
Biology Department, Boston University, Boston, Massachusetts 02215, USA.
J Am Chem Soc. 2004 Mar 24;126(11):3402-3. doi: 10.1021/ja038540u.
Fructose 1,6-bisphosphate aldolase, a glycolytic enzyme, catalyzes the cleavage of fructose 1,6-bisphosphate, resulting in two three-carbon products. The reaction of the class I enzymes, which utilize a Schiff-base intermediate, requires that the hexose be in the open-chain form. This form comprises only 1-2% of the sugar at equilibrium. The chemical form of the substrate that binds to aldolase and begins the catalytic cycle has not been unequivocally demonstrated. Transient-state kinetics in single-turnover experiments of fructose 1,6-bisphosphate with aldolase in excess reveals the rates of the intermediate steps in the cleavage reaction, including those from initial binding to Schiff-base formation. The rate of hexose Schiff-base formation was faster than the uncatalyzed rate for ring-opening of either the alpha- or beta-furanose at 4 degrees C. In addition, approach-to-equilibrium experiments reveal that aldolase binds and reacts first with 70% of fructose-1,6-bisphosphate in a fast reaction, consistent with the amount of beta-anomer in solution, and with the remaining 30%, presumably the alpha-anomer, in a slow reaction. These results indicate that aldolase must catalyze the ring-opening step and that there may be a previously unrecognized second active site on the enzyme for catalyzing this reaction.
1,6 - 二磷酸果糖醛缩酶是一种糖酵解酶，催化1,6 - 二磷酸果糖的裂解，产生两种三碳产物。I类酶的反应利用席夫碱中间体，要求己糖处于开链形式。在平衡状态下，这种形式仅占糖类的1 - 2%。与醛缩酶结合并开始催化循环的底物的化学形式尚未得到明确证实。在1,6 - 二磷酸果糖与过量醛缩酶的单周转实验中的瞬态动力学揭示了裂解反应中间步骤的速率，包括从初始结合到席夫碱形成的那些步骤。在4℃时，己糖席夫碱形成的速率比α - 或β - 呋喃糖开环的非催化速率快。此外，接近平衡的实验表明，醛缩酶在快速反应中首先与70%的1,6 - 二磷酸果糖结合并反应，这与溶液中β - 异头物的量一致，而其余30%，推测为α - 异头物，则在缓慢反应中。这些结果表明醛缩酶必须催化开环步骤，并且该酶上可能存在一个以前未被认识的第二个活性位点来催化此反应。
