| Abstract
| - Δ5-3-Ketosteroid Isomerase (KSI) catalyzes the isomerization of 5,6-unsaturated ketosteroidsto their 4,5-unsaturated isomers at a rate approaching the diffusion limit. The isomerization reaction followsa two-step general acid−base mechanism starting with Asp38-CO2- mediated proton abstraction from asp3-hybridized carbon atom, α to carbonyl group, providing a dienolate intermediate. In the second step,Asp38-CO2H protonates the C6 of the intermediate providing a 4,5-unsaturated ketosteroid. The details ofthe mechanism have been highly controversial despite several experimental and computational studies ofthis enzyme. The general acid−base catalysis has been proposed to involve either a catalytic diad or acooperative hydrogen bond mechanism. In this paper, we report our results from the 1.5 nanosecondmolecular dynamics (MD) simulation of enzyme bound natural substrate (E·S) and enzyme boundintermediate (E·In) solvated in a TIP3P water box. The final coordinates from our MD simulation stronglysupport the cooperative hydrogen bond mechanism. The MD simulation of E·S and E·In shows that bothTyr14 and Asp99 are hydrogen bonded to the O3 of the substrate or intermediate. The average hydrogenbonding distance between Tyr14-OH and O3 becomes shorter and exhibits less fluctuation on E·S → E·In.We also observe dynamic motions of water moving in and out of the active site in the E·S structures. Thisfree movement of water disappears in the E·In structures. The active site is shielded by hydrophobic residues,which come together and squeeze out the waters from the active site in the E·In complex.
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