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알코올 탈수소 효소의 K228G 위치 특이적 변이와 효소 특성

알코올 탈수소 효소의 K228G 위치 특이적 변이와 효소 특성
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Site-dercted mutagenesis of Lys-228 into Gly in horse liver alcohol dehydrogenase and the properties of K228G mutant enzyme
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대학원 약학과
알코올 탈수소 효소K228GLys-228
이화여자대학교 대학원
Lys-228 in horse liver alcohol dehydrogenase isoenzyme E was mutated to Gly-228 by phosphorothioate-based site-directed mutagenesis and the kinetic constants and the reaction mechanism of the mutant enzyme were compared to the wild-type enzyme. Comparison of the results from this study showed that all of the kinetic constants for the mutant enzyme were larger than those for the wild-type enzyme. Of particular interest were the observations that the dissociation constants for NADH and NAD^(+)(K_(iq) and K_(ia)) were increased 132 and 459 times. The product inhibition patterns suggested that the reaction mechanism catalyzed by the mutant enzyme was changed to random Bi Bi from ordered Bi Bi mechanism. And the turnover number in the forward reaction was increased 2.6 times, but that of the reverse reaction was reduced 0.2 times. While the activity of the wild-type enzyme was increasd about 10 times, that of the mutant enzyme was little affected by acetimidylation of the amino groups of lysine. The above results could attribute to the increase of coenzyme dissociation from the enzyme by modification at Lys-228 residue site of horse liver alcohol dehydrogenase.;말의 간 알코올 탈수소효소(Horse liver alcohol dehydrogenase:HLADH)의 Lys-228잔기를 glycine으로 위치특이적으로 변이시켜 얻은 K228G HLADH의 kinetic studies와 ethyl acetimidate 처리에 이한 효소 활성의 변화에 대한 결과는 다음과 같았다. 1. K228G HLADH isoenzyme E(HLADH-E)과 K228 wild-type HLADH-E의 kinetic studies를 실시하였을 때의 각각의 kinetic constant는 ◁표 삽입▷(원문을 참조하세요) a: NAD^(+), b: ethanol, p: acetaldehyde, q: NADH, TN_(f): turnover number of forword reaction, TN_(r): turnover number of reverse reaction과 같았다. 2. Product inhibition studies를 실시하였을 때 반응기전이 order Bi Bi 형식에게 ramdom Bi Bi 형식으로 바뀌었음을 알 수 있었다. 이로부터 조효소와 수소결합을 형성함으로써 조효소 부착, 이탈에 관여하는 Lys-228이 수소결합을 형성할 수 없는 glycine으로 바뀜으로 해서 조효소의 이탈이 용이하게 되었음을 추정할 수 있었다. 3. K228G HLADH-E와 wild-type HLADH-E에 ethyl acetimidate 처리를 하였을 때 wild-type에서는 약 10배의 활성 증가를 보인 반면, K228G에서는 활성에 별로 영향을 받지 않은 것으로 보아 Lys-228이 효소의 활성에 관여함을 알 수 있었다.
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