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

• Main Authors: Yi-Hsun Chang, 張藝薰
• Other Authors: Chi-Ching Hwang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/98601101131610947073

博士 === 高雄醫學大學 === 醫學研究所 === 98 === 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.