| Summary: | 碩士 === 高雄醫學大學 === 生物化學研究所 === 100 === The substrate binding loop (T188-M213) of 3a-hydroxysteroid Dehydrogenase /carbonyl reductase (3a-HSD/CR) of the short chain dehydrogenase/reductase superfamily Comamonas testosteroni, situated between the βF sheet and the aG helix, appearred an important variable substructure . The reversible oxidation-reduction of 3a-HSD/CR catalyzed androsterone and NAD+, formed androstanedione and NADH. A previous study showed that binding NADH with S114A mutant led to the conformational change, which could be observed by the circular dichroism measurement and fluorescence spectrum. When the enzyme was binded with NAD+, it induced the combination of quality changes on the conformation ring, prompted T188 and the NAD+ nicotinamide formation of hydrogen bonds. In addition, we observed that distance between hydroxyl group of S114 and carbonyl group of P185 located near the substrate binding loop increased by 0.19 A in the the enzyme-NAD+ binary complex. In this thesis, we characterized the roles of P185 and T188 in the substrate binding loop by site-directed mutagenesis, circular dichroism, fluorescence spectrum, and kinetic studies to probe the conformational changes. Mutants of T188A, T188S, T188W, P185A, P185G, P185L, P185W, and double mutants of W173F/T188W, W173F/P185W, W173F/I206W, and W173F/L197W 3a-HSD/CRs were generated and characterized. 3a-HSD/CR is ordered Bi Bi catalytic mechanism. Kinetic experiments showed that the T188 and P185 enzyme catalytzed group mutations on the catalytic reaction. Compared to wild-type, mutants of T188A increased the Michaelis constant Km for androsterone, inhibition dissociation constant (KiNAD) by 8.7 and 5.5 folds respectively, which meaned the T188 instead of alanine, broke NAD+ and substrate bad combination of hydrogen bonds on the ring T188 and catalytic constants kcat. T188A mutant caused the increase of the dissociation of nucleotide, thereby increasd the rate of releasing product. In the condition of of NAD+ fixed at 5mM, and change androsterone concentration of mutant enzyme activity, the impact of T188A and T188S mutant enzymes on kcat/Km decreased by 4.7 and 3.9 folds, and kcat increased by 3.6 and 7.6 folds; for T188S, Km increased to 81; for T188W and the double mutant W173F/T188W, kcat/Km decreased by 1.3 × 105 and 3.8 × 103 folds respectively, and kcat decreased by 3 × 104 and 2.0 × 103 folds respectively. The multiply decrease of enzyme activity suggested the importance of T188 in the catalytic reaction. Based on proline more rigid of the amino acid, the impact of single enzyme P185A and P185G mutations on kcat/Km decreased by 15 and 5 folds, and kcat decreased by 2.7 and 2.1 folds; for P185L, kcat/Km decreased by 667 folds and kcat decreased by 24 folds; for P185W, kcat/Km decreased by 1.7 × 103 folds while kcat decreased by 619 foldd. Thus, the amino acid mutations had enhanced the catalytic loop of the swing. For the double mutant W173F/P185W, its impact on kcat/Km, decreased by 3.8 × 103 folds and on kcat deceased by 1.8 × 103 folds. Finally, for W173F/I206W its impact on kcat/Km and kcat increased by 416.7 and 15.9 folds,while W173F/L197W its impact on kcat/Km decreased by 8 folds and kcat decreased by 3 folds.
CD spectra showed that there was no sigifinicant changes on secondary strcture of the T188 mutant enzyme of 3α-HSD/CR, while the P185 mutant enzymes caused changes in secondary structure. Adding NADH and androsterone, which formed binary and ternary complex, the conformation was not further affected. Monitoring pH10.5 environment by the fluorescence of the 295nm stimulate endogenous enzyme, the highest wavelength of the mutants W173F/T188W, W173F/P185W, W173F/I206W, W173F/L197W were offset from 330nm to 348 nm, 347 nm, 349 nm , and 349nm respectively. This showed that the substrate binding loop was located on the T188 and P185 hydrophilic (red shift) environment. Furtherly observing the dissociation constant Kd of the binding of enzyme and NAD+ by the fluorescence titration method, T188 mutant enzyme increased 4-6.5 folds, while the P185 mutant enzymes increased 1-3.9 folds. This suggested that the mutated enzyme surely affected the binding loop and swing functions.
Taking these results, in the process of 3a-HSD/CR acting on steroid hormone metabolism and the reversible redox reaction catalysis of androsterone and NAD+, P185 mutant enzymes plays a key role in the substrate binding loop conformaiton and while the T188 mutant enzymes in cofactor binding and catalysis.
|
|---|
