Protein Structure and Catalytic Mechanism of Amino Acid Racemase

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1. The following results have been obtained by the research on glutamate racemase. (1) Glutamate racemase has been purified from cell extracts of a lactic acid bacterium, Pediococcus pentosaceus, and its enzymological properties and reaction mechanism have been analyzed. The enzyme catalyzed an internal proton transfer under single turnover conditions with a deuterium-labeled substrate or deuterium oxide as the solvent. (2) The gene coding for glutamate racemase has been cloned into Escherichia coli. An efficient method was established for purification of the enzyme from the clone cell extracts. (3) A convenient procedure has been developed for the enantioselective synthesis of various D-amino acids by a multi-enzyme system including glutamate racemase and D-amino acid transaminase.2. The following results have been obtained by the research on alanine racemase. (1) The structural gene coding for thermostable alanine racemase of a mesothermophilic bacterium, Bacillus stearothermophilus, … More has been cloned and expressed in E. coli. The enzyme was purified to homogeneity from cell extracts of E. coli carrying a plasmid designated pICR4. The alanine racemase gene sequenced was found to contain an open reading frame of 1158 nucleotides. The molecular weight of the enzyme subunit was estimated to be 43,341. (2) The alpha-helical and beta-structure contents were calculated to be about 34 and 26%, respectively, from CD data. CD measurements of the denaturation process of enzyme by guanidine hydrochloride showed the presence of a stable intermediate during the denaturation. It has been suggested that the enzyme subunit is composed of two structurally dissimilar domains connected by a short polypeptide. (3) A synthetic DNA fragment has been inserted into the alanine racemase gene at the position corresponding to the hinge region. The gene was expressed in E. coli to produce an active enzyme with a molecular structure similar to the wild-type enzyme. (4) Single crystals of alanine racemase were prepared by the polyethylene glycol vapor diffusion method. X-ray crystallographic analysis is currently in progress. Less