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Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of
Abstract:
The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Electron paramagnetic resonance (EPR) spectroscopy reveals the active site structure of manganese(II) diphosphate (ADP) complex in yeast 3-phosphoglycerate kinase. The enzyme selectively binds the alpha configuration of the Mn(II)ADP complex, with water molecules and a charged oxygen ligand in the metal
Area of Science:
- Biochemistry
- Enzymology
- Spectroscopy
Background:
- 3-phosphoglycerate kinase is a crucial enzyme in glycolysis.
- Understanding the active site structure is key to enzyme mechanism studies.
- Manganese(II) (Mn(II)) is often used as a paramagnetic probe for metal-binding sites in enzymes.
Purpose of the Study:
- To investigate the structural and stereochemical properties of the Mn(II)ADP complex at the active site of yeast 3-phosphoglycerate kinase using EPR spectroscopy.
- To determine the coordination environment of Mn(II) and the binding selectivity of the enzyme for ADP stereoisomers.
Main Methods:
- Electron paramagnetic resonance (EPR) spectroscopy was employed to study Mn(II) complexes with ADP and 3-phosphoglycerate kinase.
- The use of regiospecifically 17O-labeled ADP and H2 17O allowed for detailed analysis of the Mn(II) coordination sphere.
- EPR spectra of complexes with (Rp)- and (Sp)-[α-17O]ADP epimers were compared to assess stereochemical selectivity.
Main Results:
- EPR data indicate Mn(II) is coordinated to the α- and β-phosphate groups of ADP, with additional ligands including negatively charged oxygen and 2-3 water molecules.
- The enzyme exhibits stereoselective binding, preferentially accommodating the (Rp)-α-configuration of the Mn(II)ADP complex.
- Addition of 3-phosphoglycerate did not alter the EPR spectrum, while vanadate addition caused significant spectral changes.
Conclusions:
- Yeast 3-phosphoglycerate kinase selectively binds the (Rp)-α configuration of the Mn(II)ADP complex at its active site.
- EPR spectroscopy provides valuable insights into the coordination chemistry and stereochemical preferences of enzyme-metal-nucleotide complexes.
- The active site structure is sensitive to the binding of substrates and inhibitors like vanadate.