人類粒腺體蘋果酸酶活性中心腺苷三磷酸結合位置之探討

Abstract

人類粒腺體蘋果酸酶 ( EC 1 . 1 . 1. 39 ) 催化氧化脫羧反應是以二價金屬離 子作為輔因子，催化蘋果酸分解為丙酮酸及二氧化碳，並且伴隨著將輔酶 NAD(P)+ 還原成為 NAD(P)H。且其活性會受到反丁烯二酸和腺苷三磷酸的調控，屬於異位調節酶；反丁烯二酸為蘋果酸酶活化劑而腺苷三磷酸為其抑制劑。在結晶結構上顯示，蘋果酸酶是由四個完全相同單元體所組成的四聚體，每個單元體中含有兩分子的 NAD+，分別位於活性中心及異位調節區；另外酶與腺苷三磷酸複合物的晶體結構則顯示了腺苷三磷酸的結合位置與NAD+ 的相同。因腺苷三磷酸亦可於活性中心佔據 NAD+ 的結合位置而可能直接影響酶活性。本研究設計利用針對腺苷三磷酸於活性區相關位置 ( R165、N259及E314 ) 利用定點突變的方式，改變為其他胺基酸，配合酶動力學，探討腺苷三磷酸對人類粒腺體蘋果酸酶可能造成的活性影響以及結構變化。結果顯示，腺苷三磷酸確實會抑制酶活性對，對酶之受質蘋果酸及 NAD+ 競爭均屬於競爭性抑制現象，於 Km,NAD 酶動力學方面，重組野生型、R165A、 N259A及E314A 無反丁烯二酸存在與有反丁烯二酸存在下，上升的倍率無明顯的差異，只有R165A的差別較大；E314A 無論有無反丁烯二酸存在，其 Km,NAD都較重組野生型蘋果酸酶降低7點多倍；於Km,malate 中，R165A 無論在無反丁烯二酸或是有反丁烯二酸存在下，數值並不會有所改變，此種情形有別於重組野生型及其餘突變株；在 Km,Mg 方面，重組野生型及三種突變株，無反丁烯二酸存在的情形之下，是有反丁烯二酸存在下 1.5-2 倍。腺苷三磷酸抑制實驗中，R165A 與 N259A 受腺苷三磷酸抑制情形較重組野生型輕微，而 E314A 受抑制情形反而較重組野生型多，顯示了此三種突變位置確實有可能是與腺苷三磷酸是重要結合位，因此突變後產生了不同的情形；而在抑制常數（Ki）實驗方面，R165A、 N259A皆較重組野生型大，而 E314A Ki,malate 與Ki,NAD皆較重組野生型降低2倍，分別代表了不同的意義。

Human mitochondrial NAD(P)+-dependent malic enzyme (EC 1.1.1.39, mNAD-ME) catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with the concomitant reduction of NAD(P)+ to NAD(P)H. The enzyme also requires divalent cation, Mn2+ or Mg2+, as an essential cofactor. The catalytic activity is allosterically regulated by fumarate as an activator and by ATP as an inhibitor. The enzyme is homotetrameric protein with four monomers arranged in the corners of the planar. The first solved crystal structure of mNAD-ME in complex with NAD+ and has two NAD+ molecule bound per monomer. These two sites are designated as the active site and allosteric site, respectively. Subsequently the structure of mNAD-ME-ATP complex has also be solved, that the binding site of ATP is in the active site as well as in the allosteric site. We used site-directed mutagenesis to mutate three residues in the ATP-binding site (R165、N259 and E314). We examined the function properties of the mutants by kinetic analysis. The results show that the Km value for NAD of R165A and N259A (Km,NAD) are the similar to those for wild-type whether fumarate presence or not, but the Km,NAD value of E314A is decreased by 7-fold. In the part of the Km value for malate (Km,malate), the Km,malate of R165A is not different between fumarate presence and absence, it indicated fumarate can not directly activate the R165A mutate enzyme. In the part of the Km value for Mg2+ (Km,Mg2+), the value of absence of fumarate is higher 1.5-2 fold than the value of presence of fumarate for wild type enzyme and three mutated enzyme. In ATP inhibited study, the enzyme activity of E314A was inhibited by ATP, which was weaker then wild type, but R165A and N259A are opposite to wild type. It suggests that three positions are important for ATP-binding. In the aspect of Ki study, overall of the Ki value for R165A and N259A is higher than wild type enzyme, but the Ki,malate and Ki,NAD of E314A are decrease than wild type about 2-fold. These represent difference significance constructions, respectively.