臺灣博碩士論文加值系統

　　在Corynebacterium glutamicum中，prephenate dehydratase是苯丙胺酸生合成途徑中主要酵素之一，此酵素會受到終產物（phenylalanine）之迴饋抑制。利用PCR任意點突變法及功能性互補試驗篩選抗mFP(m-fluorophenylalanine)之突變株，經胺基酸序列比對，得知Ser-99為共同之變異點。將Ser-99進行一系列定點突變分析，測定其酵素活性及動力學分析，Ser-99-Cys及Ser-99-Met變異酵素之活性仍保有野生型prephenate dehydratase之70%，在反應液中加入50μM之phenylalanine時，Ser-99-Cys之活性完全喪失，而Ser-99-Met之活性降至20%，其Ki值比原來提升5倍。推測Ser-99在迴饋抑制調節及蛋白質結構上扮演了重要的功能。
　　比對各物種prephenate dehydratase之胺基酸序列的保留區域（conserved region），歸納出可能會影響prephenate dehydratase活性的相關胺基酸殘基，針對此些胺基酸殘基進行定點突變（site-directed mutagenesis），其中Glu-64改變成valine, aspartate, glutamine及serine後，Glu=64-Asp保有25%之酵素活性，其餘者均已喪失活性。經動力學分析得知，Glu-64-Asp之Km值提高4.5倍，Kcat值也提升1.7倍，催化效率降低至野型的40%。推測此Glu-64之carboxyl group在酵素反應中參與nucleophile reaction，以協助催化反應的進行。
　　將Thr-183分別改變成alanine, serine及tyrosine, Ala-183及Tyr-183會失去酵素活性，而Thr-183-Ser活性剩餘4%。酵素動力學分析結果，Km值提高7倍，Kcat值只有野生型的9%，至於催化效率為原來的1.3%。推測此Thr-183可能藉由氫鍵與基質形成交互作用。將Phe-185分別改變為leucine及tyrosine, Phe-185-Leu變異酵素完全失去活性，Phe-185-Tyr的活性降為野生型的3%。動力學分析結果發現，Phe-185-Tyr之Km值為野生型的26倍，Kcat值降低至10%，而催化效率只有野生型的0.4%。推測Phe-185以"non-covalent force"方式與基質作用。綜合上述之研究，顯示出Thr-183與Phe-185在酵素反應中，扮演與基質結合的作用。

　　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.