Studies on the Functional Role of Serine 114 in the Catalytic Mechanism and Acid-Base Catalysis of 3Alpha-Hydroxysteroid Dehydrogenase

• Main Authors: Hsien-Hua Hsu, 徐樺
• Other Authors: Chi-Ching Hwang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/88398311909450513732

碩士 === 高雄醫學大學 === 醫學研究所 === 91 === 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