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Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2004 (English)In: Analytical Biochemistry, ISSN 0003-2697, Vol. 332, no 2, 203-214 p.Article in journal (Refereed) Published
Abstract [en]

The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.

Place, publisher, year, edition, pages
Elsevier , 2004. Vol. 332, no 2, 203-214 p.
Keyword [en]
Catalysis, Chymotrypsin/metabolism, Cytomegalovirus/*enzymology, Enzymes; Immobilized/chemistry/metabolism, Kinetics, Protein Conformation, Research Support; Non-U.S. Gov't, Serine Endopeptidases/*chemistry/*metabolism, Surface Plasmon Resonance/*methods
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-7290DOI: 10.1016/j.ab.2004.06.008PubMedID: 15325287OAI: oai:DiVA.org:uu-7290DiVA: diva2:131080
Available from: 2007-10-31 Created: 2007-10-31Bibliographically approved
In thesis
1. Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes: Applications for Antiviral Drug Development
Open this publication in new window or tab >>Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes: Applications for Antiviral Drug Development
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs.

The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors.

Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors.

The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding.

Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.

Place, publisher, year, edition, pages
Uppsala: Institutionen för naturvetenskaplig biokemi, 2005. 56 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 51
Biochemistry, SPR biosensor, HIV-1 reverse transcriptase, HCMV protease, interaction kinetics, drug discovery, non-nucleoside inhibitor, resistance, Biokemi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-5797 (URN)91-554-6250-2 (ISBN)
Public defence
2005-05-31, Room B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2012-04-13Bibliographically approved

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