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Metalloenzymes

Metalloenzymes. Kinases Catalyze the transfer of a phosphoryl group from ATP to various substrates Substrate + MgATP ―> phospho-substrate + MgADP All kinases require Mg(II) to form a complex with ATP Some kinases require additional metal ions for substrate binding. Dehydrogenases

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Metalloenzymes

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  1. Metalloenzymes Kinases Catalyze the transfer of a phosphoryl group from ATP to various substrates Substrate + MgATP ―> phospho-substrate + MgADP All kinases require Mg(II) to form a complex with ATP Some kinases require additional metal ions for substrate binding Dehydrogenases Catalyze redox reactions using either NAD(P) or FAD Reduced substrate + NAD(P) ―> oxidized substrate + NAD(P)H Reduced substrate + FAD ―> oxidized substrate + FADH2 Most dehydrogenases are not metalloenzymes

  2. Arginine Kinase arginine + MgATP ―> phosphoarginine + MgADP MgATP arginine The Mg ion binds to the terminal phosphate groups of ATP and helps stabilize the negative charges during phosphoryl transfer

  3. Creatine KinaseNMR structure creatine + MgATP ―> phosphocreatine + MgADP This reaction allows the “storage” of high energy phosphate groups in muscle tissues transition state complex NMR relaxation studies positioned the Mn(II) ion across the α- and β-phosphate groups of ADP Nitrate binds as a planar analog of the phosphoryl group in the transition state

  4. Creatine KinaseX-ray structure transition state complex A high resolution structure of the transition state complex shows that the Mg actually bridges between the β-phosphate and the nitrate group bond angles and distances Mg(II) does not coordinate to any protein groups The remaining donor atoms come from solvent waters Acta Cryst.D63, 381 (2007)

  5. Pyruvate kinaseNMR structure pyruvate + MgATP ―> phosphoenolpyruvate + MgADP NMR studies have established the spatial relationships between the metal ions and substrates in addition to the Mg(II) needed to bind to ATP this enzyme requires two other metal ions another divalent metal ion binds the phosphorylated substrate and a monovalent cation is also involved in substrate binding

  6. Pyruvate KinaseX-ray structure The enzyme will bind both Mn2+ and K+ even in the absence of ATP

  7. Protein kinases protein + MgATP ―> phosphoprotein + MgADP There are a large number of protein kinases with specificities for different target proteins Protein phosphorylations are one of the most common control mechanisms to regulate the activities of proteins Once again MgATP is the phosphoryl donor, with the divalent metal ion coordinated to the phosphate groups

  8. Nucleoside diphosphate kinase NDP + MgATP ―> NTP + MgADP This enzyme is responsible for transferring high energy phosphate groups between different nucleotide pools

  9. Nucleoside diphosphate kinase Active site structure of NDP kinase complex with MgADP A terminal phosphate has been modeled into the structure and the position of Mg shifted by 1 Å Model of the transition state for phosphoryl transfer from ATP to His122 Mg ion and Arg92 help neutralize the negative charge on the phosphoryl group

  10. Alcohol Dehydrogenase alcohol + NAD ―> aldehyde + NADH Zn(II) binds at the active site adjacent to the alcohol binding site A second bound zinc plays a structural role, stabilizing the enzyme J. Molec. Biol. 235, 777 (1994)

  11. Alcohol DehydrogenaseZinc binding sites catalytic Zn site structural Zn site Catalytic zinc sites typically have an open ligand position (which may be occupied by a water) to allow interactions between the metal ion and the substrate

  12. Alcohol Dehydrogenaseactive site structures native Zn enzyme Cd enzyme with bound alcohol DMSO bound but not coordinated DMSO directly coordinated to Cd

  13. Threonine dehydrogenase threonine + NAD ―> 2-amino-3-oxobutanoate + NADH The product then decarboxylates to aminoacetone (A) Loss of activity upon incubation with a metal ion chelator (B) correlation between residual enzyme activity and Zn(II) content Thus, it appears that threonine dehydrogenase contains a single bound metal ion that is essential for catalytic activity

  14. Threonine DehydrogenaseX-ray structure Each subunit contains two bound zinc ions The buried Zn plays a structural role The catalytic Zn at the active site can be removed and replaced

  15. Sorbitol dehydrogenase sorbitol + NAD ―> D-fructose + NADH room temp. low temp. From these EXAFS data the proposed Zn coordination site consists of a cysteine and 4 additional oxygen donor atoms

  16. Sorbitol DehydrogenaseX-ray structure zinc coordination Zn(II) is coordinated by one sulfur, one nitrogen, and two oxygen atoms in a distorted tetrahedron This coordination is unaffected by the binding of NAD However, the binding of an inhibitor does affect Zn(II) coordination Structure11, 1071 (2003)

  17. Isocitrate Dehydrogenasemultinuclear NMR studies isocitrate + NADP ―> α-ketoglutarate + CO2 + NADPH This is a critical step in carbohydrate metabolism that produces a reduced coenzyme to drive biosynthetic redox reactions 113Cd NMR 25Mg NMR • Cd(II) + isocitrate • Cd-ICDH • Cd-ICDH + NADP analog • Mg(II)-ICDH • Mg(II)-ICDH + isocitrate The binding of NADP and isocitrate each cause broadening of the NMR signals This indicated that these substrates are bound in close proximity to the metal ion

  18. Isocitrate Dehydrogenasemetal ion effects Several divalent metal ions, including Mg, Cd and Ca, can bind to the enzyme However, not all metal ions are equally effective in supporting catalytic activity Mg(II) and Ca(II) have comparably binding affinities to the enzyme But the Ca(II) enzyme form is essentially inactive This is presumably due to the different size and geometric requirements for Ca(II) vs. Mg(II)

  19. Isocitrate DehydrogenaseX-ray structure The active site is located between the two protein domains

  20. Isocitrate Dehydrogenaseactive site structure While NADP has several phosphate groups that can potentially bind to the metal ion, in fact the metal is found coordinated directly to the isocitrate substrate • Role of Mn(II) • Electrostatic binding to isocitrate (metal bridged mode) • Facilitate decarboxylation

  21. Isocitrate Dehydrogenasehuman cytosolic enzyme Ca(II) binding site 7-coordinate pentagonal bipyramidal carboxyl & hydroxyl groups of isocitrate Asp252, Asp275, Asp279 & a water molecule While the Ca(II) ion binding in the same location as does Mg(II), the different in coordination geometry does not allow catalysis to occur J. Biol. Chem.278, 36897 (2003)

  22. Summary • All kinases require Mg(II) to coordinate to the ATP phosphate groups • pyruvate kinase requires additional divalent and monovalent cations for substrate binding • NDP kinase contains a structural Ca(II) site • alcohol, threonine, and sorbitol dehydrogenases are each zinc metalloenzymes • isocitrate dehydrogenase uses a Mg(II) ion to facilitate a decarboxylation reaction

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