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 Mg 2+ and many divalent transition metal ions Strong Lewis acid Also true for many transition metal ions (Mg 2+ and Ca 2+ are much weaker) No redox chemistry Fairly high stability of complexes (see Irving- Williams series) Reasonably fast ligand exchange rates d 10  no CFSE: facile changes in geometry and coordination numbers Good bio-avaliabilty (today)

4. 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 Proteins tune the properties of metal ions

5. 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 > 10 11 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 pK a is lowered. The polarised or even deprotonated water then acts as strong base/nucleophile to attack an electrophilic centre 2. Polarising bound substrate for attack by base ++ -- OR’ (for esterases) can also be e.g. NHR’ (peptidases)

8. Peptide bond hydrolysis mechanism Using polarised water as nucleophile R-NH 2 + R 2 -COOH

9. Structures

10. carboxypeptidase A Peptidases/proteases Thermolysin

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

12. A classic: Carbonic anhydrase First protein recognised to contain zinc CO 2 + H 2 O HCO 3 - + H + One of the most efficient enzymes known (acceleration of reaction by factor of 10 7 ) Three histidine ligands Fourth site occupied by H 2 O, OH - Crucial for carbon fixation in photosynthetic organisms and for respiration in animals and man

22. 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

23. 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)

24. Tripodal ligands Often only a single relevant binding conformation

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

26. 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

27. Macrocyclic ligands as scaffolds The low pK a of 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  Has been estimated at 35 (water 80)

28. 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

29. 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