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Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2006 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 398, no 3, 393-398 p.Article in journal (Refereed) Published
Abstract [en]

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the K-D was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and > 100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pK(a) values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate-and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.

Place, publisher, year, edition, pages
2006. Vol. 398, no 3, 393-398 p.
Keyword [en]
active conformation, affinity, calcium binding, human macrophage elastase, matrix metallopeptidase-12 (MMP-12), pH-dependency
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-95763DOI: 10.1042/BJ20051933ISI: 000240793800008PubMedID: 16737445OAI: oai:DiVA.org:uu-95763DiVA: diva2:170098
Available from: 2007-04-19 Created: 2007-04-19 Last updated: 2011-06-15Bibliographically approved
In thesis
1. Protease Activity, Inhibition and Ligand Interaction Analysis: Developments and Applications for Drug Discovery
Open this publication in new window or tab >>Protease Activity, Inhibition and Ligand Interaction Analysis: Developments and Applications for Drug Discovery
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present study has focused on characterising protease-ligand interactions in the context of drug discovery. The proteases that have been studied are human matrix metallopeptidase 12 (MMP-12), HIV-protease and Hepatitis C virus (HCV) NS3/NS4A protease. These studies have involved kinetic characterisation of protease-inhibitor interactions using biosensor technology, as well as determination of inhibition and activity regulation by using activity assays.

The regulation of MMP-12 activity by calcium was proposed, based on the study of the calcium dependence of MMP-12 activity. Furthermore, it was shown that the high affinity of hydroxamate-based inhibitors of MMP-12 were due to slow dissociation of the enzyme-inhibitor complex by using a new biosensor assay for the study of interactions between MMP-12 and ligands.

A study of the pH-dependency of protease-inhibitor interactions revealed that the interaction kinetics of HIV-protease inhibitors differed with pH in a way that could be related to the inhibitor structures. This suggested that the forces of interaction are different in the association and dissociation phases of an interaction. Furthermore, it demonstrated the usefulness of pH as a variable in characterising protein-ligand interactions.

Results applicable in the discovery of drugs against Hepatitis C were obtained, with the analysis of structure-activity relationships of novel inhibitors. Furthermore, the mode of binding imposed by key functional groups of the inhibitors was explored by investigating the effect of pH on the interactions with NS3.

The results show the importance of using appropriate model systems for drug discovery by selecting relevant targets and assay conditions. Furthermore, the usefulness of kinetic rate information in drug discovery is demonstrated. Thus, by contributing to the knowledge of protease-ligand interactions, applicable to both protease inhibitor interactions and protease activity regulation, this thesis is expected to have an impact on the field of protease inhibitor development and drug discovery in general.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 294
Keyword
Biochemistry, proteases, kinetics, biosensors, inhibition, calcium, drug discovery, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-7822 (URN)978-91-554-6866-8 (ISBN)
Public defence
2007-05-11, B7:113a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2007-04-19 Created: 2007-04-19 Last updated: 2017-05-04Bibliographically approved

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Danielson, Helena

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