T-44、Y-118及K-131在人類雙羥擬脂醯胺去氫酶反應機制中所扮演的角色

T-44、Y-118與K-131在人類雙羥擬脂醯胺去氫酶 反應機制中所扮演的角色 陳雅慧 中山醫學大學 營養系 台中市建國北路一段110號 摘要 雙羥擬脂醯胺去氫酶（dihydrolipoamide dehydrogenase; E3）是嘌呤核苷雙硫氧化還原酶家族（pyridine nucleotide disulfide oxidoreduc- tase family）其中成員。此族群主要特徵是具有活性鹼基（active base）、活性雙硫基（active disulfide）和FAD，其活性中心可在雙硫基受質（dithiol substrate）與接受者（NAD+或NADP+）之間傳遞電子。 酮酸去氫酶複合體位於粒腺體(mitochondria)內，是由酮酸去氫酶(α-keto acid dehydrogenase; E1)、雙羥擬脂醯胺醯基轉移酶(dihydro- lipoamide acyltransferase; E2)及雙羥擬脂醯胺去氫酶(dihydrolipoamide dehydrogenase; E3)等三種酵素依特定比例所共同組成的複合體。該複合體可將酮酸(α-keto acid)氧化脫羧產生acyl CoA、CO2及NADH，故酮酸去氫酶複合體在醣類、脂肪酸及胺基酸的中間代謝上扮演重要的角色。 在酮酸去氫酶複合體中E1和E2具受質專ㄧ性(substrate speci- ficity)，然而E3是此酵素複合體的共同組成部份，所以當E3發生缺陷時，三種酮酸去氫酶複合體的活性均會受到影響，因此E3在能量代謝上扮演很重要的角色。 本實驗主要探討是有關於人類雙羥擬脂醯胺去氫酶所飾演的角色，當位於T-44、Y-118與K-131之胺基酸發生突變時，其蛋白質的表現、純化以及特徵是否受到影響。經由蛋白質純化分析表發現酵素比活性(specific activity)比Wild-typeE3 : T44S : Y118E : K131R為100 : 28.5 : 37.5 : 31.5 ;相對的FAD含量比Wild-typeE3 : T44S : Y118E : K131R為100 : 71.3 : 87.9 : 57.7。 再經分子量測定皆仍維持同質雙體(homodimer)，約為102 kDa ;酵素正反應之代謝迴轉速率(kcat)分別為E3之79.2 %(T44S)、50.5 % (Y118E)與31.3 %(K131R)，而逆反應之代謝迴轉速率(kcat)則分別為E3之32.9 %(T44S)、29.8 %(Y118E)與80.9 %(K131R)，且T44S和Y118E與受質DHL、NAD+兩者之Km皆比E3低。 另外，由氧化還原中點電位測定，結果發現T44S與Y118E之FAD還原中點電位分別為-290 mV與-270 mV皆顯著上升(E3為-314 mV)。綜合上述結果推測T-44、Y-118與K-131主要是穩定FAD結合區的結構以及參與FAD氧化還原作用。

The Role of T-44,Y-118 and K-131 in the Reaction Mechanism of Human Dihydrolipoamide Dehydrogenase(E3) Ya-Hui Chen Department of Nutritional Science,Chungshan Medical University,No.110,Sec.1,Chien-Kuo N.Rd.,Taichung City,Taiwan,R.O.C. ABSTRACT Dihydrolipoamide dehydrogenase (E3) belongs to the enzyme family of pyridine nucleotide-disulfide oxidoreductases and catalyzes the trans- fer of electrons from dihydrolipoamide to FAD cofactor , then to the NAD+. E3 is also the common com ponent of α-ketoacid dehydrogenase complexes including pyruvate dehydrogenase (PDC) , branched-chain α-ketoacid dehydrogenas complexes and α-ketoglutarate dehydrogenas complexes. A deficiency in E3 leads to the deficiency of all these three α-ketoacid dehydrogenase complexes. E3 plays an important role in the energy metabolism indeed. To investigate the reaction mechanism of human dihydrolipoamide dehydrogenase (E3), three mutant human E3s, T44S, Y118E and K131R, were overexpressed, purified and characterized. The specific activities of mutant proteins are 28.5%, 37.5% and 31.5 % to that of the wild-type E3. The FAD content analysis indicated that these three mutant E3s have about 71.3 %, 87.9% and 57.7% of FAD content compared to that of wild-type E3. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetics data demonstrat- ed that the kcat of forward reaction of mutant proteins were decreased to about 79.2% (T44S), 50.5% (Y118E) and 31.3% (K131R). The kcat of reverse reactions were decreased to about 32.9% (T44S), 29.8% (Y118E) and 80.9 % (K131R) compared to that of wild-type E3.In T44S and Y118E the Km of DHL and NAD+ were lower than E3. The mild-point oxidation-reduction potential of mutant proteins were increased from -314 mV (E3) to -290 mV (T44S) and -270 mV (Y118E). It suggests T-44, Y-118 and K-131 involved in FAD binding and oxidoreduction of FAD during the reaction. Keywords: Dihydrolipoamide Dehydrogenase, site-directed mutagenesis, enzyme kinetics.