Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169
AbstractAspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni2+. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production. View Full-Text
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Min, W.; Li, H.; Li, H.; Liu, C.; Liu, J. Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169. Int. J. Mol. Sci. 2015, 16, 28270-28284.
Min W, Li H, Li H, Liu C, Liu J. Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169. International Journal of Molecular Sciences. 2015; 16(12):28270-28284.Chicago/Turabian Style
Min, Weihong; Li, Huiying; Li, Hongmei; Liu, Chunlei; Liu, Jingsheng. 2015. "Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169." Int. J. Mol. Sci. 16, no. 12: 28270-28284.