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Zinc enzymes

Zinc enzymes. Lecture 7: Catalytic zinc Lewis acidity, substrate orientation and polarisation. Questions we will aim to answer in this session: Why zinc ?. Why zinc and not any other metal ? In reactions where zinc acts as an acid catalyst: why zinc and not just another acid ?

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Zinc enzymes

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  1. Zinc enzymes Lecture 7: Catalytic zinc Lewis acidity, substrate orientation and polarisation

  2. Questions we will aim to answer in this session: Why zinc ? • Why zinc and not any other metal ? • In reactions where zinc acts as an acid catalyst: why zinc and not just another acid ? • How do proteins tune the properties of metal ions such as zinc ? • Why it is difficult to mimick protein function with small molecule complexes ?

  3. Relevant properties • Small ionic radius: 0.65 Å • Highly concentrated positive charge (unmatched by organic acids) • But same is true for Mg2+ and many divalent transition metal ions • Strong Lewis acid • Also true for many transition metal ions (Mg2+ and Ca2+ are much weaker) • No redox chemistry • Fairly high stability of complexes (see Irving-Williams series) • Reasonably fast ligand exchange rates • d10 no CFSE: facile changes in geometry and coordination numbers • Good bio-avaliabilty (today)

  4. Proteins tune the properties of metal ions • Basic concept: The same metal ion can perform different tasks • Depending on environment generated by the protein • Simple: Opening of coordination sites for substrate: Structural Zn has 4 protein ligands, catalytic Zn usually only 3 • Presence of at least 2 Cys ligands (large thiolate sulfurs) reduces likelyhood for expansion of coordination sphere

  5. Proteins tune the properties of metal ions • Co-ordination number: • The lower CN, the higher the Lewis acidity • Co-ordination geometry • Proteins can dictate distortion • Distortion can change reactivity of metal ion • Weak interactions in the vicinity: second shell effects • Hydrogen bonds to bound ligands • Hydrophobic residues: dielectric constant can change stability of metal-ligand bonds

  6. Zinc enzymes (see Table 11) • Mono- or polynuclear • Zinc overwhelmingly bound by His, and carboxylates (Asp and Glu) • Some have also Cys • Stability: K usually > 1011 M-1 • Most catalytic zinc sites have only 3 protein ligands • One free site for substrate or coordinated water/OH- • Most prominent: Hydrolytic enzymes

  7. Hydrolysis mechanisms: Exploiting Lewis acidity 1. Bound water is polarised, and pKa is lowered. The polarised or even deprotonated water then acts as strong base/nucleophile to attack an electrophilic centre d- d+ 2. Polarising bound substrate for attack by base OR’ (for esterases) can also be e.g. NHR’ (peptidases)

  8. Peptide bond hydrolysis mechanismUsing polarised water as nucleophile R-NH2 + R2-COOH

  9. Structures

  10. Peptidases/proteases carboxypeptidase A Thermolysin

  11. Same protein ligands: Glu, His, Hisdifferent coordination number and geometry Thermolysin Carboxypetidase A

  12. A classic: Carbonic anhydrase • First protein recognised to contain zinc • CO2 + H2O HCO3- + H+ • One of the most efficient enzymes known (acceleration of reaction by factor of 107) • Three histidine ligands • Fourth site occupied by H2O, OH- • Crucial for carbon fixation in photosynthetic organisms and for respiration in animals and man

  13. Synthetic models of enzymes • Only a few years ago, elucidating structures of proteins was very difficult • In order to understand metalloproteins, small-molecule complexes were synthesised to mimic the behaviour of the protein-bound metal • More amenable to structural, spectroscopic and mechanistic studies • Structural models: To model the ligand sphere; can compare spectroscopic properties (relevant for Fe and Cu) • Functional models: much more difficult; trying to mimic reactivity

  14. Synthetic models • Not trivial to mimic enzyme sites: • Proteins provide rigid scaffold, define coordination sphere: often distorted tetrahedra • In small complexes, higher coordination numbers (5 and 6) are common, usually less distorted • Often formation of bi- or polynuclear complexes • Successful Examples: • Pyrazolylborates and other tripodal ligands (Parkin, Vahrenkamp, ...) • Macrocycles (Kimura, Vahrenkamp, ...) • Calix-arenes (Reinaud)

  15. Tripodal ligands • Often only a single relevant binding conformation

  16. A case study: dipicolylglycine as tripodal ligand CO Di-bromo Di-aquo Ahmed Abufarag and Heinrich Vahrenkamp, Inorg. Chem. 1995,34, 2207-2216.

  17. Better: Tris-pyrazolylborates • Bulky substitutents in 3 position of pyrazoles sterically enforce tetrahedral coordination Rainer Walz, Michael Ruf, and Heinrich Vahrenkamp, Eur. J. Inorg. Chem. 2001, 139-143

  18. Macrocyclic ligands as scaffolds • The low pKaof bound water can be achieved in small molecule complexes • Crucial: coordination number !! • CN 6: ca. 10 • CN 5: 8-9 • CN 4: < 8 • Further influence in proteins: dielectric constant e • Has been estimated at 35 (water 80)

  19. Calix-arenes • Calix[6]arene mimics the hydrophobic interior of proteins and can function like a substrate-binding pocket Seneque et al., J. Am. Chem. Soc., 2005, 127 (42), pp 14833–14840

  20. Summary • Zinc has unique properties that neither any other metal ion nor any organic compound can match: • Extremely high Lewis acidity • No redox chemistry • Flexible coordination numbers and geometry • Fast ligand exchange rates • Capabilities of zinc are influenced by protein environment

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