Chemical and Biochemical Studies on Enzyme-Catalyzed Asymmetric Decarboxylation

芳基丙二酸脱羧酶(AMDase)是我们从一株嗜碱杆菌中分离到的。它催化双取代芳基丙二酸酯的不对称脱羧基反应，得到具有光学活性的相应乙酸酯。它是一种非常独特的脱羧酶，因为它不需要其他脱羧酶和转羧酶所需要的辅酶，如生物素、辅酶A和ATP。该酶由240个氨基酸组成，包括4个半胱氨酸残基。通过抑制实验表明，这些半胱氨酸残基中至少有一个是酶活性所必需的。定点突变发现Cys188位于活性部位，那么半胱氨酸是如何激活底物的呢？结合部位的其他氨基酸残基如何与底物的官能团相互作用？使用精心设计的抑制剂进行的物理化学研究表明，半胱氨酸残基与底物形成硫醇酯键。硫醇酯基团的较大电负性被认为是通过带负电荷来稳定过渡态。此外，一些特定底物的动力学揭示了底物在活性中心的构象。酶与底物之间的CH-pi相互作用将是结合力之一。我们挑战通过随机突变来扩大底物的特异性。虽然我们还不能分离出底物特异性扩大的突变体，但已经发现了一些比野生型酶更活性的突变体。三级结构的X射线分析目前正在进行中。

Arylmalonate decarboxylase (AMDase) was isolated by us from a bacterium Alcaligenes bronchisepticus. It catalyzes asymmetric decarboxylation of disubstituted arylmalonates to give the optically active corresponding acetates. It is a very unique decarboxylation enzyme since it requires no coenzymes, such as biotin, coenzyme A,and ATP,which other decarboxylases and trancarboxylases do.This enzyme consists of 240 amino acids, including four cysteine residues. Through the inhibition experiments, it was suggested that at least one of these cysteine residues is essential for the enzyme activity. Site-directed mutagenesis revealed that Cys188 is the one that is located in the active site.Then, how does the Cys activate the substrates? How do the other amino acid residues in the binding site interact with the functional groups of the substrates? Physicochemical studies using well-designed inhibitors suggested that Cys residue forms a thiol ester bond with the substrates. Large electronegativity of thiol ester group is estimated to stabilize the transition state with a negative charge. Also kinetics of some specified substrates shed light on the conformation of the substrate in the active site. CH-pi interactions between enzyme and the substrate will be one of the binding forces. We challenged to widen the substrate specificity by random mutation. Although, we could not isolate a mutant of which substrate specificity had been widened, there were found a few mutants that were more active than the wild-type enzyme.X-ray analysis for tertiary structure is now in progress.