Chemical and Biochemical Studies on Enzyme-Catalyzed Asymmetric Decarboxylation

酶催化不对称脱羧的化学和生化研究

基本信息

批准号：
07459023
负责人：
OHTA Hiromichi
金额：
$ 4.1万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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.

芳基乳核脱羧酶（AMDase）由我们从细菌alcalium alcaligenes支气管抑制剂中分离出来。它催化了非卵巢芳基甲酯的不对称脱羧化，从而产生了光学活跃的相应醋酸盐。这是一种非常独特的脱羧酶，因为它不需要辅酶，例如生物素，辅酶A和ATP，其他脱羧酶和trancar羧酸酶也不需要。这些酶由240个氨基酸组成，包括四个囊这氨酸残基。通过抑制实验，建议至少其中一种半胱氨酸残基对于酶活性至关重要。定点诱变表明Cys188是位于活性位点的诱变。然后，Cys如何激活底物？结合位点中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的物理化学研究表明，Cys残基与底物形成硫醇酯键。据估计，硫醇酯组的大电负性可以用负电荷稳定过渡态。一些指定底物的动力学也阐明了活性位点中底物的构象。酶与底物之间的CH-PI相互作用将是结合力之一。我们挑战以通过随机突变扩大底物特异性。虽然，我们无法隔离一个扩大底物特异性的突变体，但发现一些突变体比野生型酶更活跃。x射线对高等结构的分析现在正在进行中。