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The catalytic power of ketosteroid isomerase investigated by computer simulation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2002 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 52, 15728-15735 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2002. Vol. 41, no 52, 15728-15735 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-90165DOI: 10.1021/bi026873iPubMedID: 12501201OAI: oai:DiVA.org:uu-90165DiVA: diva2:162419
Available from: 2003-03-12 Created: 2003-03-12 Last updated: 2013-05-31Bibliographically approved
In thesis
1. Computational Studies of Enzymatic Enolization Reactions and Inhibitor Binding to a Malarial Protease
Open this publication in new window or tab >>Computational Studies of Enzymatic Enolization Reactions and Inhibitor Binding to a Malarial Protease
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enolate formation by proton abstraction from an sp3-hybridized carbon atom situated next to a carbonyl or carboxylate group is an abundant process in nature. Since the corresponding nonenzymatic process in water is slow and unfavorable due to high intrinsic free energy barriers and high substrate pKa s, enzymes catalyzing such reaction steps must overcome both kinetic and thermodynamic obstacles.

Computer simulations were used to study enolate formation catalyzed by glyoxalase I (GlxI) and 3-oxo-Δ5-steroid isomerase (KSI). The results, which reproduce experimental kinetic data, indicate that for both enzymes the free energy barrier reduction originates mainly from the balancing of substrate and catalytic base pKas. This was found to be accomplished primarily by electrostatic interactions. The results also suggest that the remaining barrier reduction can be explained by the lower reorganization energy in the preorganized enzyme compared to the solution reaction. Moreover, it seems that quantum effects, arising from zero-point vibrations and proton tunnelling, do not contribute significantly to the barrier reduction in GlxI. For KSI, the formation of a low-barrier hydrogen bond between the enzyme and the enolate, which is suggested to stabilize the enolate, was investigated and found unlikely. The low pKa of the catalytic base in the nonpolar active site of KSI may possibly be explained by the presence of a water molecule not detected by experiments.

The hemoglobin-degrading aspartic proteases plasmepsinI and plasmepsin II from Plasmodium falciparum have emerged as putative drug targets against malaria. A series of C2- symmetric compounds with a 1,2-dihydroxyethylene scaffold were investigated for plasmepsin affinity, using computer simulations and enzyme inhibition assays. The calculations correctly predicted the stereochemical preferences of the scaffold and the effect of chemical modifications. Calculated absolute binding free energies reproduced experimental data well. As these inhibitors have down to subnanomolar inhibition constants of the plasmepsins and no measurable affinity to human cathepsin D, they constitute promising lead compounds for further drug development.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 52 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 816
Theoretical chemistry, enzyme mechanism, enolate, molecular dynamics, empirical valence bond, glyoxalase I, ketosteroid isomerase, triosephosphate isomerase, malaria, aspartic protease, plasmepsin, linear interaction energy, drug design, Teoretisk kemi
National Category
Theoretical Chemistry
Research subject
Molecular Biotechnology
urn:nbn:se:uu:diva-3335 (URN)91-554-5554-9 (ISBN)
Public defence
2003-04-04, B42, Biomedical Centre, Uppsala, 13:15 (English)
Available from: 2003-03-12 Created: 2003-03-12 Last updated: 2010-01-14Bibliographically approved

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