Function of amino acid sites in the substrate entryway of alanine racemase TtAlr from Thermoanaerobacter tengcongensis

Abstract:

[Objective] In order to study the function of amino acid sites A172 and S173 in the substrate entryway of alanine racemase TtAlr from Thermoanaerobacter tengcongensis MB4. [Methods] The mutant vectors were constructed by site-directed mutagenesis PCR using plasmid pET-TtAlr as the template and expressed in E. coli BL21(DE3). The enzymes were purified by affinity chromatography. D-amino acid oxidase coupling method was used to detect enzyme activity and stability of each mutant and wild type TtAlr proteins. [Results] Both TtAlr and mutant proteins were expressed and purified successfully. Results of enzymatic properties show that A172 site mutation could improve the catalytic activity of TtAlr, but the stability of enzyme proteins decreased significantly. Likewise, S173 site mutation could increase the optimal temperature of TtAlr, prolonged the half-life of the enzyme and improved its stability, but the catalytic activity of the enzyme decreased significantly. [Conclusion] A172 and S173 amino acids residues in the substrate entryway of alanine racemase TtAlr were the key sites that played a major role in the catalytic activity and stability of the enzyme protein.