title: The catalytic power of ketosteroid isomerase investigated by computer simulation.
abstract: Ketosteroid isomerase (KSI) catalyzes the isomerization of Delta(5)-3-ketosteroids and Delta(4)-3-ketosteroids at very high rates. Here we examine the principles underlying the catalytic efficiency of KSI by computer simulations using the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. The simulations reproduce available kinetic and structural data very well and allow us to examine several features of the catalytic mechanism in detail. It is found that about 60% of the rate enhancement is due to stabilization of the negatively charged dienolate intermediate by hydrogen bonding. The critical H-bond between Tyr16 and the intermediate is found to be a normal ionic H-bond with the preferred proton location on the tyrosine residue. The remaining 40% of the catalytic effect originates from a reduction of the reorganization energy of the reaction. The possibility of an active site water molecule occupying the empty cavity adjacent to the catalytic base (Asp40) is also addressed. The existence of such a water molecule could explain how the enzyme manages to maintain a low pK(a) for the general base residue.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden.
authors.forename: Isabella
authors.initials: I
authors.lastname: Feierberg
authors.forename: Johan
authors.initials: J
authors.lastname: Aqvist
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D004795:Enzyme Stability; D008956:Models, Chemical; D008958:Models, Molecular; D016958:Pseudomonas putida / Q000201:enzymology; D012997:Solvents; D055672:Static Electricity; D013251:Steroid Isomerases / Q000737:chemistry*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D012997:Solvents; D013251:Steroid Isomerases