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Geometric and Electrostatic Changes in the KSI Reaction

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posted on 22.02.2013, 08:49 by Daniel A Kraut, Paul A Sigala, Brandon Pybus, Corey W Liu, Dagmar Ringe, Gregory A Petsko, Daniel Herschlag

(A) Mechanism of KSI catalyzed isomerization of 5-androstene-3,17-dione (substrate) to 4-androstene-3,17-dione (product). In the first step a general base, Asp40, removes a proton from the steroid to form a dienolate intermediate (via a dienolate-like transition state), which receives hydrogen bonds from an oxyanion hole consisting of Tyr16 and protonated Asp103. In the second step of the reaction the steroid is reprotonated at a different position to give the product.

(B) Geometric changes accompanying the first half of the KSI reaction. Oxygen is shown in red, carbon in grey, hydrogen in blue. The ring geometry changes in the transition state and in the intermediate, becoming more planar in the intermediate. The sp2-hybridized carbonyl oxygen in the substrate becomes a predominantly sp3-hybridized oxyanion. Structures were generated using CaCHE 4.93 (Fujitsu, Tokyo, Japan) MOPAC PM5 geometry optimization [150] and rendered using CS Chem3D Pro 5.0 (CambridgeSoft, Cambridge, Massachusetts, United States).

(C) Electrostatic changes at the carbonyl group accompanying the first half of the KSI reaction. The larger “δ−” refers to increased negative charge on the oxygen atom as the reaction proceeds. The dienolate-like transition state is expected to be between the substrate and dienolate intermediate in charge arrangement, but closer to the high-energy intermediate.

(D) Schematic depiction of the steroid equilenin bound at the KSI active site. Equilenin geometrically and electrostatically resembles the dienolate reaction intermediate and transition state.

(E) Schematic depiction of a single-ringed phenolate bound at the active site of KSI D40N, the mutant enzyme used for this work. The Asp40Asn mutation mimics the protonated aspartate found in the intermediate and equilenin complexes, see (A) and (D), and leads to tighter binding of phenolate and other intermediate analogs [69, 71].




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