Section 6: Zinc based metalloenzymes

Question 1

Why are we studying zinc based metalloenzymes?

Answer 1

Second most abundant trace element in humans

• found in many essential enzymes
• has applications in catalytic processes, structural engineering of proteins and in regulation processes

Question 2

What chemical properties make Zn(II) important in biological processes

Answer 2

1. Strong Lewis acid
2. Not redox reactive
3. Ready formation of low coordinate binding sites(>acidity?)
4. Accesible coordination numbers of 4,5 and 6
5. Easily deformed coordination geometry
6. Easily undergoes mono-ligand substitution

Question 3

What is the form of zinc in biological systems?

Answer 3

Always in Zn(II) form

closed d10 configuration –> diamagnetic and colourless complexes.

Question 4

Zinc and water: a unique relationship

Answer 4

A unique feature of all characterised active mononuclear zinc site, is a water ligand, which can be activated by ionization, by polarization or be poised for displacement by a substrate
Zn-OH2 +A- –> Zn-OH +HA (ionisation)
Zn-OH2 +L –> Zn-L + H2O (displacement)
Zn-OH2 +B- –> Zn-OH +HA (polarisation

Question 5

What effect does zinc have on the polarization of water?

Answer 5

pKa of water usually =15
[Zn (H20)6]2+ =10
ZnL3(H20) =7

Zn lowers the pKa of water
Lower coordination numbers at the zinc centre also lower the pKa of the attatched H2O

Question 6

What issues are there with the study of zinc based metallocenes

Answer 6

-Its transparency
-Most Zn enzyme studies have been done by replacing Zn(II) with Co(II) which has a similar ionic radius and can tolerate similar coordination environments
-It is possible to replace the metal without great peturbation of the protein conformation.
Co(II) is a d7 metal and hence has peaks in the UV spectrum, which can then be used to monitor coordinate geometry and pH studies.

Question 7

How mechanistically is Zn replaced?

Answer 7

PICTURE HERE

Question 8

Example 1: CA II -what is it

Answer 8

Carbonic anhydrase II
-A metalloenzyme found in red blood cells
- catalyses the reversible hydration of CO2 which is fundamental for removal of CO2 from active metabolizing sites- without catalyst occurs very slowly
-rate increases by factor of 10^7
CO2 + H2 <=> HCO3- + H+

Question 9

What is the structure of CA II

Answer 9

Zinc atom at bottom of a 15 A cleft in the metalloenzyme

H2O-Zn-(N(His))3

Question 10

Mechanism of CA II

Answer 10

Picture

Question 11

Does the peptide environments around the active site effect the catalytic activity

Answer 11

yes, surrounding peptide groups are often nvolved in the mechanism either acid/base or stabilisation etc, all which speed up the reaction

Question 12

What is CPA?

Answer 12

Carboxypeptidase A
-a digestive enzyme found in the pancreas
-catalyses the cleavage of a peptide link in a polypeptide chain and helps organisms to assimilate(digest) the proteins that are ingested
Picture

Question 13

What are some limitations for the CPA reaction

Answer 13

The cleavage only occurs at the C-terminal amino acid and has a high selectivity for the substrates that have a C-terminal amino acid containing large aliphatic or Ph substituents 

Question 14

CPA structure

Answer 14

Large MW,
307 amino acids and one zinc atom
near surface of the protein is a pocket containing zinc atom with two N(His) and one bidenate O(glu)
guanidium ion of Arg and carboxylat group of Glu also involved

Question 15

Proposed mechanism of CPA diagram

Answer 15

PICTURE