zinc enzymes lecture 7: catalytic zinc lewis acidity, substrate orientation and polarisation
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Zinc enzymes
Lecture 7: Catalytic zinc
Lewis acidity, substrate orientation and polarisation
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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 ?
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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)
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• 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
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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
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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
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Hydrolysis mechanisms: Exploiting Lewis acidity
OH2Zn
OHZn H
++
Zn2+
OR'
R
O OH- Zn2+
OR'
R
O OH
n
Zn2+
R'OH RCOOH+ + +
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
2. Polarising bound substrate for attack by base
+-
OR’ (for esterases) can also be e.g. NHR’ (peptidases)
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Peptide bond hydrolysis mechanismUsing polarised water as nucleophile
R-NH2 + R2-COOH
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Structures
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carboxypeptidase A
Peptidases/proteases
Thermolysin
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Same protein ligands: Glu, His, His
different coordination number and geometry
Carboxypetidase A Thermolysin
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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
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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
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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)
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Tripodal ligands
• Often only a single relevant binding conformation
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A case study: dipicolylglycine as tripodal ligand
Di-aquo
CO
Di-bromo
Ahmed Abufarag and Heinrich Vahrenkamp, Inorg. Chem. 1995,34, 2207-2216.
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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
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Macrocyclic ligands as scaffolds
• The low pKa 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)
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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
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Summary
• Zinc has unique properties that neither any other metal ion nor any organic compound can match:Extremely high Lewis acidityNo redox chemistryFlexible coordination numbers and
geometryFast ligand exchange rates
• Capabilities of zinc are influenced by protein environment