利用定點突變方法改變人類絲胺酸消旋酶及絲胺酸脫水酶之酵素功能

Abstract

絲胺酸消旋酶主要功能是產生D-絲胺酸，D-絲胺酸是在腦部中的N-甲基,D-天門冬胺受體 (NMDARs)之共促效劑 (co-agonist)，哺乳類動物的絲胺酸消旋酶可將L 及D-絲胺酸互相轉換，也可將L 及D-絲胺酸脫水轉變成丙酮酸。我們觀察到絲胺酸消旋酶和人體中另一個酵素絲胺酸脫水酶在蛋白質序列上只有23 %的相似性但結構上相似度很高，但絲胺酸脫水酶卻只有一種將L-絲胺酸轉變成丙酮酸的功能。它們之間的關係引發了我們高度興趣。在蛋白質序列比對分析中,指出在絲胺酸消旋酶催化活性中心的第84 號絲胺酸相對位置於絲胺酸脫水酶催化活性中心的第65 號丙胺酸。此兩酵素活化反應皆需要輔因子Pyridoxal 5’phophate (PLP)，在沒有受質存在情況下會和酵素上的離胺酸形成Schiff base，當受質存在時會取代離胺酸和PLP 形成Schiff base。為了要深入探討兩酵素之間的關係，我們將絲胺酸消旋酶的第84 號絲胺酸突變成丙胺酸，結果發現其完全失去消旋作用，僅存在可將L-絲胺酸轉變成丙酮酸的功能。反之思考若將絲胺酸脫水酶中的第65 號丙胺酸突變成絲胺酸，是否可以使其增加消旋作用的活性。結果顯示其能夠增加利用D-絲胺酸轉變成丙酮酸的功能，因為原本是丙胺酸無法成為催化鹼去攻擊D-絲胺酸在Cα 上的氫原子，當其成為絲胺酸時即可成為催化鹼，額外增加利用D-絲胺酸為受質的功能。而為了能夠更加了解其作用機制，我們解出了胺酸脫水酶第65 號丙胺酸突變 成絲胺酸的結構。從活性分析及結構資料中，我們了解到絲胺酸消旋酶的第84 號絲胺酸所在的方位對於在消旋作用以及D-絲胺酸利用中扮演很重要的角色，並提供了對其作用機制更深入的了解。

Serine racemase catalyzes the production of D-serine, a co-agonist of the NMDARs in the brain. Mammalian serine racemase is involved in the reversible conversion of L- to D-serine, as well as the dehydration activity toward L- and Dserine. We observed human serine racemase gene shows 23% identity with that of the human serine dehydratase (SD), which catalyzes the dehydration of L-serine to yield ammonia and pyruvate. Sequence alignment shows that the corresponding residue Ala65 in the human serine dehydratase is aligned with the catalytic Ser84 in the human serine racemase. One such mutant protein is a serine to alanine substitution at residue 84, located at the active site of human serine racemase. The S84A mutation caused the loss of isomerization activity and D-serine dehydratase of serine racemase. Whereas it retained the capability to act as an L-serine dehydratase activity. The single mutant of human serine dehydratase A65S protein increased 5-fold D-serine dehydratase activity at pH=9. To improve our understanding of the relationship between human serine racemase and human serine dehydratase mechanism, we have determined the X-ray crystal structure of the human serine dehydratase A65S mutant protein at 1.54Å resolution. Our results show that the S84 residue in human serine racemase, proximity to the substrate in an ideal orientation, plays an important role in shuttling the proton required for isomerization. The biological activity analysis of target mutagenesis and useful structural information have paved the way for mechanistic studies and have provided a framework for interpretation of those results.