Nucleotide Binding to Human Ump-cmp Kinase Using Fluorescent Derivatives -- a Screening Based on Affinity for the Ump-cmp Binding Site.
From: Laboratoire d’Enzymologie Moléculaire et Fonctionnelle, FRE 2852 CNRS-Paris 6, Institut Jacques Monod, Paris, France.
The FEBS journal
- Publish Date: Jul 2007
- ISSN: 1742-464X
- Volume: 274
- Issue: 14
- Pages: 3704-14
- Medium: Print
- Language: English
- Citation (JAMA): Topalis Dimitri, Kumamoto Hiroki, Amaya Velasco Maria-Fernanda, et al. Nucleotide Binding to Human Ump-cmp Kinase Using Fluorescent Derivatives -- a Screening Based on Affinity for the Ump-cmp Binding Site.. FEBS J. Jul 2007;274:3704-14
Abstract
Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.
Mesh Headings (Keywords): Animals, Anthranilic Acids, Binding Sites, Biological Products, Cytidine Diphosphate, Cytidine Monophosphate, Fluorescent Dyes, Humans, Kinetics, Models, Molecular, Nucleoside-Phosphate Kinase, Protein Structure, Tertiary, Spectrometry, Fluorescence, Substrate Specificity, Uridine Monophosphate
Check for Full Text / PubMed Unique Identifier (PMID): 17608725
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