Studies on the Functional Role of Lysine 159 in the Catalysis of 3 alpha-Hydroxysteroid Dehydrogenase/Carbonyl Reductase

• Main Authors: Zhen Wei Li, 李振雄
• Other Authors: Chi-Ching hwang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/04764582190412382063

碩士 === 高雄醫學大學 === 生物化學研究所 === 91 === 3a-Hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR) from Comamonas testosteroni, catalyzes the oxidoreduction at position 3 of steroids and carbonyl reduction of a nonsteroidal aldehydes and ketones, and uses the NAD+ as the cofactor, androsterone as the substrate. The 3a-HSD/CR gene is 774 base pair long, and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. Base on previous studies, a homology search revealed that amino acid sequence highly conserved in the short chain dehydrogenase/reductase (SDR) superfamily are present in 3a-HSD/CR. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment essential for catalytic activity of SDR protein. The functional groups involved in the catalysis were proposed based on the crystal structure and mutagenesis studies. The amino acid residues of Tyr, Ser, and Lys act as a catalytic triad to facilitate the oxidative-reductive reaction in the Comamonas testosteroni 3a-HSD. It was purposed that the Tyr functions acts as the catalytic base, whereas Ser stabilizes the substrate and Lys lowers the pKa of the Tyr-OH. The residue Tyr acts the general base to remove the proton from hydroxyl group of substrate and facilitates the hydride transfer during the catalysis. To know the functional role of Lys 159 in the catalysis of 3a-HSD, enzyme was expressed, purified and mutated. Kinetic analysis, pH profile and isotope effects are studied to elucidate the enzyme-catalyzed reaction. The results indicated that the Km is increase 11-fold, kcat decreased 28-fold and kcat/Km drcreased 315-fold in mutant K159A. The pKa in K159A shifted to 9.1 compared to wild type of 7.7. The observed isotope effects wrer increased with mutant of D(V/K)=1.9, DVmax=2.3 compared to wild type of D(V/K)=1.3, DVmax=1.17. The results indicated that Lys lowers the pKa of the Tyr-OH, and facilitates the catalytic reaction.