- 學位論文
3a羥基類固醇脫氫酶絲胺酸114在催化反應機構及酸鹼催化作用中所扮演功能之探討
Studies on the Functional Role of Serine 114 in the Catalytic Mechanism and Acid-Base Catalysis of 3Alpha-Hydroxysteroid Dehydrogenase
摘要
從Comamonas testosteronin﹙原稱為Pseudomonas testosteronin﹚所分離出的3a羥基類固醇脫氫酶﹙3alpha-hydroxysteroid dehydrogenase﹐3a-HSD﹐EC 1.1.1.50﹚，屬於短鏈脫氫酶家族﹙short chain dehydrogenase﹐SDR﹚的成員之一，其對於類固醇激素的生合成及調節扮演著相當重要的角色。3a羥基類固醇脫氫酶為NAD(P)依賴性的酵素，以高度的部位特異性（regiospecificity）及立體特異性（stereospecificity）催化類固醇羥基及酮基間的氧化還原反應。3a羥基類固醇脫氫酶與其它短鏈脫氫酶家族成員間的序列相同性相當低，通常只有10-30％，但在立體結構方面卻表現出高度相似性的a/b摺疊結構。在3a羥基類固醇脫氫酶的活化位置上具高度保留性之由serine﹙絲胺酸﹚, tyrosine﹙酪胺酸﹚及lysine﹙離胺酸﹚三個殘基組成catalytic triad﹙分別是Ser-114、Tyr-155及Lys-159﹚，其中尤其以tyrosine為最高度保留的位置。 之前曾有研究提出catalytic triad中絲胺酸的功能，主要是穩定基質與酵素間的結合之作用，並幫助催化反應中酪胺酸共同進行質子傳遞。為了進一步瞭解絲胺酸在3a羥基類固醇脫氫酶催化反應機構中所扮演的角色，所以選擇catalytic triad中的絲胺酸來做進一步的探討。本實驗是利用定點突變方法將活化位置上的絲胺酸改變成丙胺酸，藉由動力學上的分析、pH profile及同位素效應，來探討3a羥基類固醇脫氫酶活化位上Ser-114在催化機構所扮演的角色。從定位突變置換的酵素動力學結果得知，突變型S114A之kcat比野生型減少了800倍，kcat/Km降低了2143倍，Km增加了近3倍。在pH-profile的研究中，野生型酵素的pKa值為7.7±0.1，而突變型S114A之pKa值為7.39±0.08，結果和野生型相當。以氫負離子轉換的androsterone-3-d當作受質，皆能測得到野生型和突變型﹙S114A﹚之同位素效應。在獲得動力學結果DV為1.11±0.03及DV/K為1.08±0.04的暸解下，顯示rate-limiting step主要是在hydride transfer的過程中。根據pH-profile的結果，發現Ser-114並非扮演催化鹼的角色，而是在催化反應中穩定與受質之間的結合。
並列摘要
3Alpha-Hydroxysteroid dehydrogenase (3a-HSD, EC 1.1.1.50) from Comamonas testosteronin (formerly Pseudomonas testosteronin) is a member of the protein superfamily of short-chain dehydrogenases. It plays a significant role in steroid hormone metabolism and regulation. 3a-HSD, which is a NAD(P)(H)-dependent enzyme, catalyzes the oxidoreduction of hydroxyl and ketone groups of steroids with high regiospecificity and stereospecificity. The pairwise sequence identity between 3a-HSD and other short-chain dehydrogenases is low, typically 10-30%, but all available three-dimensional structures display highly similar a/b folding pattern. In the active site, 3a-HSD has a triad of catalytically important Ser-114, Tyr-155, and Lys-159 residues, of which Tyr-155 is the most conserved residue. The function of catalytic triad has been proposed that Ser stabilizes the substrate binding and helps proton relay between substrate and cofactor. In this study, the role in the catalytic mechanism of the residue Ser-114 was analyzed by a combination of site-directed replacement, pH profile, and isotope effect. Enzyme kinetic data from mutagenetic replacement for S114A is 800-fold decrease in kcat, 2143-fold lower than wild type in kcat/Km, nearly 3-fold increase in Km,androsterone. In the pH-profile study, the pKa value of S114A is 7.39±0.08, which is similar to that of wild type (pKa=7.7±0.1). Primary kinetic isotope effects were measured on the hydride transfer of androsterone-3-d for both wild type and mutant type (S114A) 3a-HSD. A finite observed kinetic parameters DV of 1.11±0.03 and DV/K of 1.08±0.04 indicate a rate-limiting step on the hydride transfer. Based on the pH-profile study, Ser-114 does not acts as the general base catalyst, and stabilize in the substrate binding
參考文獻
延伸閱讀
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