Proton Affinity of the Oxyanion Hole in the Active Site of Ketosteroid Isomerase
نویسندگان
چکیده
منابع مشابه
Proton affinity of the oxyanion hole in the active site of ketosteroid isomerase.
The absorption spectra of a series of inhibitors bound at the active site of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida were found to exhibit substantial variations in the contributions of the protonated and deprotonated forms. Systematic variation of the inhibitor solution pK(a) combined with a method of quantifying the contributions of each protonation state showed the oxyanion ...
متن کاملUsing Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site
Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of t...
متن کاملTesting geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole.
Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups a...
متن کاملDissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole.
The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 10(5)-fo...
متن کاملEvaluating the catalytic contribution from the oxyanion hole in ketosteroid isomerase.
Prior site-directed mutagenesis studies in bacterial ketosteroid isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side c...
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ژورنال
عنوان ژورنال: Biochemistry
سال: 2010
ISSN: 0006-2960,1520-4995
DOI: 10.1021/bi100074s