Download Bio-Inorganic Chemistry

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 ?
• How do proteins tune the properties of metal
ions such as zinc ?
• Why it is difficult to mimick protein function
with small molecule complexes ?
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 IrvingWilliams series)
• Reasonably fast ligand exchange rates
• d10  no CFSE: facile changes in geometry and
coordination numbers
• Good bio-avaliabilty (today)
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
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
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
Hydrolysis mechanisms:
Exploiting Lewis acidity
Zn
OH
OH2
+
Zn
H
+
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
Zn
2+
dO
OR'
d+ + OHR
Zn
2+
O
OR'
OH
R
Zn
2+
+
R'OH+ RCOOH
n
2. Polarising bound substrate for attack by base
OR’ (for esterases) can also be e.g. NHR’ (peptidases)
Peptide bond hydrolysis mechanism
Using polarised water as nucleophile
R-NH2
+
R2-COOH
Structures
Peptidases/proteases
carboxypeptidase A
Thermolysin
Same protein ligands:
Glu, His, His
different coordination number and
geometry
Carboxypetidase A
Thermolysin
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
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
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)
Tripodal ligands
• Often only a single relevant binding
conformation
A case study: dipicolylglycine as
tripodal ligand
CO
Di-bromo
Di-aquo
Ahmed Abufarag and Heinrich Vahrenkamp, Inorg. Chem. 1995,34, 2207-2216.
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
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 e
• Has been estimated at 35 (water
80)
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
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