| 저자(한글) |
Jang, Do Soo,Choi, Gildon,Cha, Hyung Jin,Shin, Sejeong,Hong, Bee Hak,Lee, Hyeong Ju,Lee, Hee Cheon,Choi, Kwan Yong |
| 초록 |
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-ketosteroi isomerase (KSI) catalyzes the allylic isomerization of ${ Delta}^5$ -3-ketosteroid to its conjugated ${ Delta}^4$ -isomers at a rate that approache 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 steroi 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. |
