Objective
Iron-containing phosphatases constitute a ubiquitous class of enzymes that are responsible for removing phosphate groups from phosphorylated proteins in a wide variety of catabolic, regulatory, and signal transduction pathways. The focus of the present proposal is upon the purple acid phosphatases, which are small, basic proteins that are highly glycosylated and contain a spin-coupled mixed-valence (Fe3+Fe2+) binuclear center at the active site; the purple color is due to a conserved tyrosine residue that coordinates to the Fe3+ site. A similar Fe3+M2+ center, lacking a tyrosine ligand, is now established for the regulatory Ser/Thr protein phophatases despite the fact that the protein structures of the two classes of enzymes are unrelated. In addition, structurally unrelated binuclear metal sites are present in a wide class of hydrolytic enzymes, including other protein phosphatases. Hence, insights derived from studies of the purple acid phosphatases are likely to be broadly applicable. The objective of the proposed research is to understand the fundamental relationship between protein and active site structure on the one hand and enzymatic and physiological function on the other in the purple acid phosphatases. No three-dimensional structural information is yet available for a mammalian purple phosphatase (although the structure of a potentially related plant enzyme has recently been reported by one of the partners), and the physiological function of the enzymes in mammals remains unresolved. This lack of understanding has thus far precluded the development of significant biotechnological applications of these enzymes, a situation that the proposed research is intended to remedy.
The proposed iron phosphatase network consists of seven groups from seven different EU countries or Associated States, and brings areas of expertise ranging from medicine to biochemistry and biophysics to inorganic chemistry to bear upon the problem. The specific objectives of the proposed research are:
(i) determination of the three-dimensional structure of at least one example of a mammalian purple acid phosphatase containing a binuclear iron center;
(ii) determination of structural and electronic parameters through the use of advanced spectroscopic studies of the active sites of these enzymes in order to address questions of active-site structure that cannot be answered by conventional X-ray structure determinations' focussing on how variations in active site structure correlate with enzymatic activity;
(iii) determination of the catalytic mechanism of the enzyme, how it is controlled by the protein structure, and how general the mechanism is within a broader class of phosphatases utilizing a binuclear metal center at the active site through application of modern molecular biology and advanced spectroscopy techniques;
(iv) elucidation of the physiological role(s) of these phosphatases in both a model eukaryote (yeast) and in a model mammal (mice).
The research is divided into four work packages, each of which combine the expertise of relevant partners to attack one of the above objectives across a broad front utilizing complementary experimental systems and techniques to ensure maximal progress. The approach focusses on three-dimensional structure determinations of the target enzymes, but also has a significant component that requires the development of new techniques for study of biological structure and function as well as the design of new enzymes with altered structure and reactivity. The results of the proposed research will facilitate a variety of new biotechnological applications of the target enzymes, including phosphate-sensitive electrodes and new phosphate detectors for use in immunochemical and/or receptor-based bioanalytical systems. Additional outcomes of the research will include the development of biomedical uses of the enzymes themselves or of specific low molecular weight inhibitors of enzyme function developed with the aid of the three-dimensional structural information to be acquired in this work in the diagnosis and treatment of autoimmune and metabolic diseases.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences zoology mammalogy
- natural sciences chemical sciences inorganic chemistry
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
- natural sciences physical sciences optics spectroscopy
- natural sciences biological sciences molecular biology
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Coordinator
1018 TV Amsterdam
Netherlands
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