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Computer simulation of primary kinetic isotope effects in the proposed rate-limiting step of the glyoxalase I catalyzed reaction
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.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2000 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 30, 22657-22662 p.Article in journal (Refereed) Published
Abstract [en]

The proposed rate-limiting step of the glyoxalase I catalyzed reaction is the proton abstraction from the C1 carbon of the substrate by Glu172. Here we examine primary kinetic isotope effects and the influence of quantum dynamics on this process by computer simulations. The calculations utilize the empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the enzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 ± 1.3 is predicted in reasonable agreement with the experimental result of about 3. Furthermore, the magnitude of quantum mechanical effects is found to be very similar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with attaining the required accuracy in order for the present approach to be useful as a diagnostic tool for the study of enzyme reactions are also discussed.

Place, publisher, year, edition, pages
2000. Vol. 275, no 30, 22657-22662 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90164DOI: 10.1074/jbc.M000726200OAI: oai:DiVA.org:uu-90164DiVA: diva2:162418
Available from: 2003-03-12 Created: 2003-03-12 Last updated: 2013-06-12Bibliographically 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.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 816
Keyword
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
Identifiers
urn:nbn:se:uu:diva-3335 (URN)91-554-5554-9 (ISBN)
Public defence
2003-04-04, B42, Biomedical Centre, Uppsala, 13:15 (English)
Opponent
Available from: 2003-03-12 Created: 2003-03-12 Last updated: 2010-01-14Bibliographically approved

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Åqvist, Johan

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