| Summary: | 碩士 === 國立清華大學 === 生物技術研究所 === 90 === In lysine biosynthesis of Escherichia coli, aspartokinase III takes part and plays a key role in the phosphorylation of aspartic acid. Aspartokinase III is a allosteric enzyme, and feedback-inhibited by lysine. The kinetic and regulatory properties of lysine-sensitive AK III are well characterized and lysine inhibition of the AK III enzyme is cooperative and noncompetitive with aspartic acid. Interactions between subunits have also been demonstrated to exhibit a ligand induced association - dissociation behavior.
Previously, a mutant lysC gene has been cloned by spontaneous mutation of a recombinant plasmid harboring a wild-type lysC gene. Nucleotide sequence determinations of the mutant lysC gene show only A to C substitution, and Thr to Pro at position 352 (T352P). After kinetic and binding studying, it shows that the point mutation implicates the alleviation of binding affinity for substrate and loss of the binding site of allosteric inhibitors, lysine and leucine.
In order to figure out the possible effect of the position 352 in AK III, the methodology of site-directed mutagenesis was taken, and the residue 352 was changed from Thr to Ala, Asp, Gly, Asn and Val. Then the changed target amino acid residue at 352 position of pUC19AK3 plasmids were transformed to Escherichia coli DH5α and expression. To isolate the five mutant AK III protein, Superdex 75 gel filtration column and Mono-Q anion exchange column were used.
In steady state initial velocity study, kinetics was done by verifying the concentration of one substrate at a fixed concentration of the other. Therefore the initial velocity could be calculated and the primary plots (double reciprocal plot) and secondary plot were constructed.
The lysine inhibition results reveal that when position 352 was substituted by hydrophobic amino acid residues, like Ala and Val, the residual activity of the two mutant AK III are much higher than those substituted by hydrophilic amino acid, like Asp, Gly and Asn. And the and values are agree with the result.
The of T352D(10.21 ) and T352N(17.32 ) are higher than other mutants, and they are also more sensitive to lysine. Comparing values, T352D and T352 G are higher and also more sensitive to lysine than T352A and T352V.
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