人類粒線體蘋果酸酶異位調節區中天門冬胺酸102對酶活性調節機制之探討

Abstract

人類粒線體蘋果酸酶 (EC 1.1.1.39) 在結構上是由四個完全相同的單體所組成之四聚體，可分為較緊密的雙聚體界面及較鬆散的四聚體界面，而每個單體都具有各自的活性中心，能將蘋果酸及NAD(P)＋在二價金屬離子(如錳、鎂離子)的催化下，進行氧化脫羧作用，形成產物丙酮酸、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，除了活性中心之外，人類粒線體蘋果酸酶在雙聚體界面處還具有異位調節區的存在，而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。本研究希望從酶動力學的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胺基酸側鏈電荷平衡的相關性，因而經物種間胺基酸序列比對後，選擇將異位調節區中側鏈原本帶負電荷之Asp102，利用定點突變的方式改變其側鏈電荷，分別突變為丙胺酸 (Ala，不帶電)、麩胺酸 (Glu，帶負電)、 離胺酸 (Lys，帶正電）後，再以酶動力學實驗，相互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Asp102經過突變而改變了其電荷性質後，在Km及kcat等基本的動力學常數方面與野生型並沒有顯著的差異，但是在Kact值及在不同濃度反丁烯二酸測得之apparent Km值的降低程度則有所不同。可以發現到，突變型人類粒線體蘋果酸酶無論是Kact值的大小或是反丁烯二酸對apparent Km值的降低程度，都比野生型要來的低，表示突變型對反丁烯二酸的活化變得較不敏感；並且在三種突變型之間，當改變的電荷性質與野生型差距愈大，會導致Kact值愈小，apparent Km值的降低程度也愈不明顯。因此我們推測，異位調節區中的第102號胺基酸側鏈所帶電荷之不同，的確會影響反丁烯二酸對人類粒線體蘋果酸酶的活化現象；並且在異位調節區中，反丁烯二酸的結合除了受到與其直接結合的胺基酸側鏈影響之外，還會被異位調節區中其他胺基酸側鏈所帶電荷之平衡程度影響。

Human mitochondrial NAD(P)+-dependent malic enzyme (EC 1.1.1.39) is a homotetrameric protein with four monomers. This homotetramer is a double dimer structure and the dimer interface is more intimately contacted than the tetramer interface. Every monomer has its own active site and catalyzes a reversible oxidative decarboxylation of L-malate to give carbon dioxide and pyruvate in the concomitant reduction of NAD(P)+ to NAD(P)H. Human mitochondrial NAD(P)+-dependent malic enzyme is an allosteric enzyme. Fumarate acts as an activator and ATP acts as a competitive inhibitor. Fumarate is bound at the dimer interface about 30 Å away from the active site, confirming that fumarate functions 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 role of Asp102 in allosteric site involving the regulatory mechanism of fumarate activation. We used site-directed mutagenesis to mutate residue 102 in allosteric site to inspect its influence of charge (D102A、D102E and D102K). In order to compare with the wild type and three mutants, we examined the functional properties by enzyme kinetic analysis. Regardless of the existence of fumarate, the Km and kcat are no notable difference between wild type and mutants. The Kact values of wild type and mutants are reducing with the increasing concentration of substrate、coenzyme and cofactor. Furthermore the decrease degree of apparent Km are very different among wild type and mutants. From the kinetic data, we suggest that Asp102 is an essential residue in activation of fumarate and the balance of electric charge in allosteric site may play an important role in the allosteric site of human mitochondrial NAD(P)+-dependent malic enzyme.