Chemical and Biochemical Studies on Enzyme-Catalyzed Asymmetric Decarboxylation

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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.