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Rather, the enhancement may be due to the sum of many different contributions including hydrogen bonds, electrostatic and hydrophobic interactions, and positioning of the substrate on the enzyme

Posted on January 15, 2022 by president2010

Rather, the enhancement may be due to the sum of many different contributions including hydrogen bonds, electrostatic and hydrophobic interactions, and positioning of the substrate on the enzyme. Click here to view.(180K, pdf) Acknowledgments This research was supported by a grant from the National R & D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1320240). 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 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 8.1C11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1 1.7C2. 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. and one from have been studied to understand the enzyme-catalyzed heterolytic C-H bond cleavage that occurs in a wide variety of biological reactions (Gerlt et al., 1991). In the reaction catalyzed by KSI, Tyr14 and Asp99 are thought to have critical functions in stabilizing a dienolate intermediate by forming LBHB or regular hydrogen bonds with the oxyanion of the intermediate (Cho et al., 1998; Kim et al., 1997a). The 1H NMR spectrum of KSI complexed with equilenin (i.e., an intermediate analogue in the reaction) shows a highly deshielded proton resonance near 17 ppm, which has been regarded as compelling evidence for the involvement of LBHB in the catalysis (Cho et al., 1999; Zhao et al., 1996; 1997). NMR spectroscopic studies combined with site-directed mutagenesis have revealed that an LBHB RGS7 can form between Tyr14 O and C3-O of equilenin in the active site of D38N (Ha et al., 2001). The strength of the LBHB in KSI has been estimated to be at least 7.1 kcal/mol by comparing the dissociation rates of the intermediate from your Y14F and the D38N mutants (Xue et al., 1991) and by measuring the proton exchange rate of the LBHB on the pH range 4.3 to 9.0 (Zhao et al., 1996; 1997). The Y14F mutation reduced KSI by a factor of 5 104 (Kuliopulos et al., 1989) but that of the D99A mutation only a factor of 5 103 (Wu et al., 1997). In addition, Y14F and D99A mutants of KSI (with this paper we quantity the residues of KSI relating to the people of KSI) are only 1/2,000 and 1/98 instances as active as the wild-type KSI (Kim and Choi, 1995; Kim et al., 1997b), respectively; this switch suggests that by forming LBHBs, Tyr14 contributes to catalysis more crucially than does Asp99. Open in a separate windowpane Fig. 1. Reaction catalyzed by ketosteroid isomerase. Androstenolone, equilenin, and estrone are analogues of substrate, intermediate, and product of KSI, respectively. The proton at C-4 is definitely transferred by Asp 38 to the part of C-6 during the isomerization reaction. Both Tyr14 and Asp99 can stabilize the intermediate by forming a hydrogen relationship with the oxyanion of the intermediate. Tyr14 is definitely hydrogen-bonded to Tyr55 that is in turn hydrogen bonded to Tyr30 in the KSI. In this study, we measured the enthusiastic difference between LBHB and the Clemizole hydrochloride ordinary hydrogen relationship in the active site of KSI. Together with the structural analyses for the hydrogen bonds involved in the Clemizole hydrochloride catalytic reaction of KSI, our NMR spectroscopic studies revealed the putative LBHB between Tyr14 O and C3-O of equilenin observed for D38N KSI was converted to an ordinary hydrogen bond from the Y30F/Y55F mutations. The conversion of the LBHB to an ordinary hydrogen bond resulted in only marginal effects on both catalytic activity of KSI and its binding affinity for the intermediate analogue. Our results suggest that the contribution of LBHB to catalysis should be only marginal compared with that of an ordinary hydrogen relationship in the active site of KSI. MATERIALS AND METHODS Materials 5-androstene-3,17-dione (5-AND), androstenolone, equilenin and estrone were purchased from Steraloids Inc. (USA). 15N-Labeled NH4Cl was purchased from Cambridge Isotope Laboratories Inc. (USA). A Superose 12 gel filtration column was purchased from Amersham Bioscience (USA). All chemicals for the buffer remedy were purchased from Sigma (USA). All enzymes for DNA manipulation were purchased from Promega (USA). Oligonucleotides were from Genotech Inc. (Korea). Site-directed mutagenesis, manifestation and purification of mutant KSIs Site-directed mutagenesis of Y115F, Y115F/D38N, Y30F/Y55F/Y115F, Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N Clemizole hydrochloride was carried out as explained previously (Kim et al., 2000). All mutations were confirmed by sequencing the entire gene of the mutant KSI. Mutant KSIs were overexpressed in BL21(DE3) (Novagen) harboring an expression.

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