Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase
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- Title
- Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase
- Authors
- Jang, DS; Choi, G; Cha, HJ; Shin, S; Hong, BH; Lee, HJ; Lee, HC; Choi, KY
- Date Issued
- 2015-05-31
- Publisher
- KOREAN SOC MOLECULAR & CELLULAR BIOLOGY
- Abstract
- Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Delta(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta(5)-3-ketosteroid to its conjugated Delta(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 O eta and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 O eta and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/36318
- DOI
- 10.14348/MOLCELLS.2015.2266
- ISSN
- 1016-8478
- Article Type
- Article
- Citation
- MOLECULES AND CELLS, vol. 38, no. 5, page. 409 - 415, 2015-05-31
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