Enzymes that hydrolyze phosphate esters (phosphatases) play a key role in energy metabolism, in metabolic regulation, and in a variety of signal transduction pathways. Iron-containing phosphatases constitute an important and only recently recognized class of phosphatases that are involved in such diverse processes as phagocytosis and bone resorption, regulation of glycogen metabolism, muscle contraction, mitosis, and hormonal control. The long-term objective of the proposed research is the determination of the structure of the metal chromophore in the iron- containing phosphatases and elucidation of its role in catalytic phosphate ester hydrolysis.
The specific aims of the proposed research are: (1) detailed characterization of the interaction of a prototypical purple acid phosphatase containing a binuclear iron center (that from bovine spleen) with substrates, phosphate, and phosphate analogs by spectroscopic methods and elucidation of its mechanism of action; (2) characterization of the metal site(s) in the mammalian regulatory protein phosphatases. Complexes of the native (diiron) form of the bovine spleen purple acid phosphatase and its mixed metal derivatives (FeZn, FeGa, FeCu) with tetrahedral oxyanions such as phosphate and molybdate and related compounds such AMP will be examined by UV-visible, resonance Raman, EPR, ENDOR, electron spin echo envelope modulation, EXAFS, 57Fe Mossbauer, and NMR spectroscopy in order to characterize the interaction of substrates and substrate analogs with dimetal center. Emphasis will be placed on examination of inhibitor complexes with the recently identified phosphoryl-enzyme intermediate and analogous ternary complexes. The residue(s) that are phosphorylated during catalysis will be identified. Finally, a major effort will be made to determine the nature of the metal binding sites in a recombinant fungal protein phosphatase 2B (calcineurin), using a combination of metal analyses, spectroscopic studies, and site-directed mutagenesis experiments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032117-11
Application #
2176442
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1993-09-01
Project End
1995-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
11
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Virginia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Orlando, J L; Zirino, T; Quirk, B J et al. (1993) Purification and properties of the native form of the purple acid phosphatase from bovine spleen. Biochemistry 32:8120-9
Averill, B A; Vincent, J B (1993) Electronic absorption spectroscopy of nonheme iron proteins. Methods Enzymol 226:33-51
Wang, Z; Ming, L J; Que Jr, L et al. (1992) 1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen. Biochemistry 31:5263-8
Vincent, J B; Crowder, M W; Averill, B A (1992) Multiple binding sites for tetrahedral oxyanion inhibitors of bovine spleen purple acid phosphatase. Biochemistry 31:3033-7
Vincent, J B; Crowder, M W; Averill, B A (1992) Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions. Trends Biochem Sci 17:105-10
Crowder, M W; Vincent, J B; Averill, B A (1992) Electron paramagnetic resonance studies on the high-salt form of bovine spleen purple acid phosphatase. Biochemistry 31:9603-8
Vincent, J B; Crowder, M W; Averill, B A (1991) Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen. J Biol Chem 266:17737-40
Vincent, J B; Crowder, M W; Averill, B A (1991) Spectroscopic and kinetics studies of a high-salt-stabilized form of the purple acid phosphatase from bovine spleen. Biochemistry 30:3025-34
Vincent, J B; Averill, B A (1990) An enzyme with a double identity: purple acid phosphatase and tartrate-resistant acid phosphatase. FASEB J 4:3009-14
Vincent, J B; Averill, B A (1990) Sequence homology between purple acid phosphatases and phosphoprotein phosphatases. Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers? FEBS Lett 263:265-8

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