Comamonas testosteroni 3alpha-羥基固醇去氫酶/羰基還原酶催化機制之探討

The study of catalytic mechanism in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni

10.6832/KMU.2009.00032

張藝薰

羥基固醇去氫酶催化機制 ； hydroxysteroid dehydrogenase ； catalytic mechanism

高雄醫學大學醫學研究所學位論文

2009年

博士

黃啟清

繁體中文

Comamonas testosteroni 3alpha-羥基固醇去氫酶/羰基還原酶 (3alpha-HSD/CR)可催化androsterone及NAD+的氧化反應而形成androstaedione及NADH。基於結構分析及序列比對，Asn-86、Ser-114、Tyr-155及Lys-159為3alpha-HSD/CR的催化位組。而本文將探討此一催化位組在3alpha-HSD/CR催化反應中所扮演的角色。 第一章節本文利用定點突變( site-directed mutagenesis )、穩定態動力學( steady-state kinetics )及 pH profile去探討Ser-114及Tyr-155在3alpha-HSD/CR催化反應中的功能。野生型及單一突變酵素(S114A、Y155F)均呈現pH-dependence現象，然而S114A/Y155F卻為pH-independence情形。Tyr作為一般鹼的角色，以Phe取代會使pH profile呈現pH-independence的現象，因此pH profile的結果意謂著可能有另一個官能基參與在Y155F突變酵素的酸鹼催化反應。由於S114A及Y155F突變酵素為pH-dependent profile而S114A/Y155F為pH-independent pattern，因此推測當Tyr-155被取代時，其鄰近的Ser-114則替代Tyr-155成為一般鹼進行催化反應。 第二章則利用定點突變、chemical rescue及溶媒同位素效應(solvent isotope effect)探討Lys-159在質子傳遞系統中的角色。以Ala及Met取代Lys-159阻礙了質子的傳遞路徑，但藉由外加質子接受體(proton acceptors)的幫助則可促使質子轉移順利進行。根據Brønsted關係圖呈現線性關係(B= 0.85 ± 0.09)及在10 mM CAPS時所觀測到4.4的溶媒同位素效應現象，顯示質子傳遞至外加質子接受體的步驟為late transition state且為速率決定步驟。此外proton inventory實驗在野生型及K159A酵素均觀測到bowl-shaped圖形得知質子由Tyr-155經ribose的2'-OH轉移至Lys-159的過渡狀態中有兩個質子參與轉移。根據以上結果得知Lys-159可作為3??-HSD/CR催化反應中質子傳遞的角色。 第三章則更進一步以穩定動力學、螢光遮蔽實驗(fluorescence quenching)及anisotropy measurements闡述Ser-114與NADH結合的情形。S114A突變酵素的V/Et下降了3100倍，然而對受質的親合性(Km)卻無顯著變化，暗示了NADH 結合在nonproductive的構形。此外NADH結合至S114A突變酵素時會誘導酵素的二級結構發生改變。以Ala取代Ser-114失去了與Pro-185之間的氫鍵作用因而誘導酵素構形發生改變，導致NADH 結合在nonproductive的構形。此一結果暗示了Ser-114的角色可能與酵素和cofactor結合時的結構維持在適當的構形有關，進而使催化反應能順利進行。 藉由對Ser、Tyr及Lys在催化機制角色上的探討，得知其在SDR酵素的催化過程中為一不可或缺的催化組。而上述探討的重要性對於SDR家族的催化功能亦提供了基礎性的了解。

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. According to the sequence alignment and structure analysis, a catalytic tetrad of Asn-86, Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed. In this study, we will explore the functional roles of catalytic tetrad in 3alpha-HSD/CR-catalyzed reaction. In the first part, we combined site-directed mutagenesis, steady-state kinetics, and pH profile to elucidate the function of Ser-114 and Tyr-155 in 3alpha-HSD/CR-catalyzed reaction. The pH profile showed a pH-dependence in wild-type, S114A and, unexpectedly in Y155F mutant enzyme. The role of Tyr has been proposed to act as a general base. This indicates another functional group is involved in the acid-base catalysis of Y155F mutant. Furthermore, pH-independence pattern was observed in S114A/Y155F mutant enzyme. The results suggest the additional role of Ser-114 is complementary catalytically with Tyr-155 to act as a general base on the reaction catalyzed by Y155F mutant. In the second part, we investigate the role of Lys-159 in the proton relay system by using mutagenesis, chemical rescue, and solvent kinetic isotope effects study. The rate of the proton transfer is blocked in the K159A and K159M mutants, but can be rescued using exogenous proton acceptors. The Brønsted relationship is linear (B= 0.85 ± 0.09) and a value of 4.4 on D2OV is observed in K159A mutant at 10 mM CAPS, indicating the proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes, suggesting a two-proton transfer in the transition state from Tyr-155 to Lys-159 via 2'-OH of ribose. These results demostrate that the role of Lys can serve as a proton shuttle in the enzyme catalysis. In the third part, the role of Ser-114 was further expanded with the cofactor binding through steady-state kinetics, fluorescence quenching and anisotropy measurements. S114A mutant enzyme decreases 3100-fold in V/Et with no apparent change in Km for substrates, indicating NADH is bound in a nonproductive mode. Furthermore, addition of NADH to S114A mutant enzyme induces a secondary structural change. Substitution of Ala for Ser-114 eliminates the hydrogen bonding interaction with Pro-185 and may cause a conformational change in a nonproductive binding of NADH. These results suggest that Ser-114 is important in maintaining the correct conformation for nucleotide binding and facilitates the reaction. In summary, Ser, Tyr and Lys are essential in the SDR catalysis through characterizing their mechanistical roles in 3alpha-HSD/CR. The significance of the above studies provides the fundamental understanding of SDR family in general.

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