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 Irving-Williams

series)• Reasonably fast ligand exchange rates• d10 no CFSE: facile changes in geometry and

coordination numbers• Good bio-avaliabilty (today)

• 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

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

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)

Peptide bond hydrolysis mechanismUsing polarised water as nucleophile

R-NH2 + R2-COOH

Structures

carboxypeptidase A

Peptidases/proteases

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

Di-aquo

CO

Di-bromo

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 • 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 acidityNo redox chemistryFlexible coordination numbers and

geometryFast ligand exchange rates

• Capabilities of zinc are influenced by protein environment