title: Contribution of a low-barrier hydrogen bond to catalysis is not significant in ketosteroid isomerase.
abstract: Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(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 Oη 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 Oη 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.
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.|Research Institute, Genexine Co., Seongnam 463-400, Korea.
authors.forename: Do Soo
authors.initials: DS
authors.lastname: Jang
authors.affiliation: Korea Research Institute of Chemical Technology, Daejeon 305-343, Korea.
authors.forename: Gildon
authors.initials: G
authors.lastname: Choi
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.|Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Cha
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Sejeong
authors.initials: S
authors.lastname: Shin
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.|Research Institute, Genexine Co., Seongnam 463-400, Korea.
authors.forename: Bee Hak
authors.initials: BH
authors.lastname: Hong
authors.affiliation: Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors.forename: Hyeong Ju
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors.forename: Hee Cheon
authors.initials: HC
authors.lastname: Lee
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004855:Equilenin / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D066244:Proton Magnetic Resonance Spectroscopy; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013251:Steroid Isomerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D004855:Equilenin
keywords: Tyr14; enzyme catalysis; ketosteroid isomerase; low-barrier hydrogen bond