| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 86 ===
Prephenate dehydratase is the first enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. This enzyme is subject to feedback inhibition by L-phenylalaine. PCR-based random utagenesis and functional complementaton were used to screen for mFP-resistant mutants. Amino acid sequence comparisons between all the mutants led to the suspicion that Ser-99 plays a critical role in the feedback regulation of prephenate dehydratase. The position at Ser-99 was modified by site-directed mutagenesis. When Ser-99 was replaced with a methionine residue, the activity of mutated prephenate denydratase was decreased to 70%. In the presence of 50 μM phenylalanine, a further decrease of activity to 20% was observed. The Ki (by phenylalanine) of Ser-99-Met prephenate dehydratase was increased by about 5-fold. The data indicated that Ser-99 is an Important residue regarding feedback regulation and also structurally essential for prephenate dehydratase activity.
Alignment of amino acid sequences of prephenate dehydratases from different microorganisms was performed to determine the conserved amino acid residues which might be involved in enzyme catalysis. The conserved amino acid residues were changed by site-directed mutagenesis. Replacement of Glu-64 with Asp resulted in a decrease of enzyme activity to 25%. The Km and Kcat values were increased by 4.5-fold and 1.7-fold respectively. The results suggested that the r-carboxyl group of Glu-64 might serve as a nucleophile in the catalytic reaction of prephenate dehydratase. Substitution of Thr-183 by either alanine or tyrosine led to the loss of prephenate dehydratase activity; however, mutated Thr-183-Ser still retained 4% enzyme activity. In addition, the apparent Km of mutated prephentae dehydratase Thr-183-Ser was increased. These results demonstrated the interaction between the hydroxyl group of Thr-183 and prephenate. Replacement of phe-185 with tyrosine decreased the specific activity to 3%. Prephenate dehydratase of Phe-185-Tyr mutant showed an increase in the Km value by about 26-fold, suggesting that Phe-185 residue might involve in substrate binding.
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