探討人類粒線體蘋果酸酶異位調節區中第59號麩胺酸對酶活性之調節機制

Abstract

人類粒線體蘋果酸酶(EC 1.1.1.39)在結構上是由兩個相同的單體之雙聚體所組成之四聚體，而每個單體都具有各自的活性中心，能將蘋果酸及NAD(P)+在二價金屬（如錳、鎂離子）的催化下，進行氧化脫羧作用，形成丙酮酸、二氧化碳及NAD(P)H；人類粒線體蘋果酸酶的活性會分別受到反丁烯二酸及腺苷三磷酸的活化及抑制，目前研究顯示反丁烯二酸為異位活化劑而腺苷三磷酸則為競爭型抑制劑。由晶體結構證實，除了活性中心外，人類粒線體蘋果酸酶在雙聚體界面處還有異位調節區的存在，反丁烯二酸所扮演的異位活化劑角色，其活化機制可能為促使四聚體之重排。本研究希望從動力學角度來探討人類粒線體蘋果酸酶之異位調節區中，反丁烯二酸的結合與相關胺基酸側鏈電荷平衡的相關性，因此我們選擇了第59號麩胺酸的位置進行定點突變，分別突變為帶有相同電荷的天門冬胺酸（Asp）、結構相似但不帶電荷的麩醯胺酸（Gln）、帶相反電荷的精胺酸（Arg）及不帶電荷的白胺酸（Leu），再以動力學的實驗，互相比較野生型及突變型的活性差異。經由實驗發現，當異位調節區中的Glu59經過突變而改變了其電荷性質後，在Km及kcat等基本動力學常數方面與野生型並沒有顯著的差異。於反丁烯二酸實驗方面，野生型在10 mM的反丁烯二酸濃度下，活化了約1.4倍，但突變型E59L及E59R皆不受反丁烯二酸所影響，顯示E59在反丁烯二酸活化作用為一重要胺基酸；而突變型E59Q及E59D則對活化劑的需求濃度異於野生型，因此我們藉由Kact及KA方面觀察E59Q及E59D與野生型之間的差異性，結果可發現突變後，不論於E59Q或E59D皆對於反丁烯二酸需求提高，亦表示其對活化劑之親合力降低，而影響到Kact、KA及αKA值明顯比野生型來的高。在反丁烯二酸異構物的實驗方面，我們想藉著結構差異處來了解活化中心或異位調節區的結合方式。經由實驗證實了Glu59在人類粒線體蘋果酸酶異位調節區中，扮演極重要之角色，即使並非直接與反丁烯二酸產生鍵結，可能藉由與Arg67或可能還包含有Lys57方面來影響反丁烯二酸的親和力。

Human mitochondrial NAD(P)+-dependent malic enzyme (EC 1.1.1.39) is a homotetrameric protein consisting of a dimer of dimers. Each monomer has its own active site and catalyzes a reversible oxidative decarboxylation of L-malate to pyruvate and CO2 in the concomitant reduction of NAD(P)+ to NAD(P)H. This reaction also requires the presence of divalent cations, Mg2+ or Mn2+ as an essential cofactor. Human mitochondrial NAD(P)+-dependent malic enzyme is an allosteric enzyme with fumarate as an activator and ATP as an inhibitor. Fumarate is bound at the dimer interface about 30 Å away from the active site, confirming that fumarate function through an allosteric mechanism. It is possible that fumarate promotes the reorganization of the enzyme tetramer, and this may be the molecular mechanism for its allosteric effects on the catalysis by human m-NAD-ME. In this thesis, we tried to elucidate the functional role of allosteric site Glu59 involving the regulatory mechanism of fumarate activation. We used site-directed mutagenesis to mutate residue 59 in allosteric site to inspect its influence of charge or polarity (E59D, E59Q, E59R, and E59L). In order to compare with the wild-type and four mutants, we examined their functional properties by enzyme kinetic analysis. Regardless of the existence of fumarate, the values of Km and kcat are no notable differences among wild-type and mutants. Our kinetic studies showed that the wild-type is activated about 1.4-fold in the presence of 10 mM fumarate with saturating concentrations of the substrates. The E59L and E59R mutant could not be activated by fumarate, indicating E59 is an important residue for fumarate activation. The E59Q and E59D mutants, which could be activated by fumarate, however, showed different tendency compared with wild-type on activator concentration. Therefore, we focus the Kact and KA values on WT, E59D, and E59Q. The Kact, KA and αKA values in E59D and E59Q were notable higher than WT, indicating the affinities of fumarate to E59D and E59Q were lower than WT. In fumarate analog experiments, we can observe their specific structure result in the different binding ability. Our data indicate that Glu59 is important for fumarate binding even though it was not direct interaction on fumarate, but it seems via the effect upon R67 or(and) K57 to change its affinity for fumarate.