Lecture 8 Peptides - Hydrolysis and Enzymes

Peptides are quite stable to ‘chemical’ conditions but are easily hydrolyzed by enzymes.
Understand the mechanism of amide bond hydrolysis for zinc ion proteases such as carboxypeptidase A.
Rationalize the binding of a substrate (peptide or drug) into an enzyme active site of a zinc ion protease using hydrogen bonding, ionic bonding, and metal coordination concepts.

Proceeds with difficulty because:
- $H_2O$ is a poor nucleophile.
- The $C=O$ of an amide is unreactive towards nucleophiles.
Can be achieved by heating with strong aqueous acid, e.g., 6 M HCl, but even then, the reaction proceeds slowly.

Protein is a necessary component of our diet.
Our metabolism must efficiently hydrolyze the peptide bonds to liberate the amino acids, especially the essential amino acids (the ones we don’t make).
This is achieved by:
- The low pH of our stomach (chemical hydrolysis of peptides is possible).
- Mostly by enzymes called proteases, that effect hydrolysis of the peptide bond.

There are many types of large proteins that are enzymes, that catalyse the cleavage of different bonds in different compounds
Washing powders can contain different types of enzymes to break down the different compounds in stains:
- Proteases cleave amide bonds.
- Lipases cleave triglycerides into glycerol and fatty acids.
- Amylases break down starches.
Washing powders usually only contain one type of enzyme, though some have two or all three.
Some people have an immune response/a reaction to enzymes in washing powder.

Many proteases hydrolyze peptides in a manner analogous to the hydrolysis of esters.
For proteases hydrolyzing peptide bonds:
- Instead of the acid ( $H^+$ ), a metal ion often polarizes the carbonyl.
- The nucleophile is often an amino acid side chain or a water molecule that has reacted with an amino acid side chain.

Different proteases are very specific in the peptide sequence and site they cleave.
Carboxypeptidase A recognizes the carboxylate of a C-terminal amino acid next to an amino acid with a non-polar side chain and efficiently hydrolyzes the adjacent peptide bond.
Thus, it effectively clips off the C-terminal amino acid.
It is an example of a metalloenzyme, with $Zn^{2+}$ playing an integral role at the active site.

The reactants are held close together; the peptide to be cleaved, the soon-to-be nucleophile, and the zinc ion are all in close proximity.
$Zn^{2+}$ makes the carbon of the $C=O$ more susceptible to nucleophilic attack.

A glutamic acid side chain (i.e., a carboxylate) reacts with the water that is coordinated to the zinc ion.
This is not something ‘chemically possible’ in a solution but is possible in an enzyme active site because of the coordination of the water to the zinc ion.
The zinc ion lowers the $pK_a$ of the water.
The hydroxide nucleophile can now attack the carbonyl carbon of the amide bond to be cleaved.
This is possible because of the zinc ion polarizing the carbonyl bond (drawing electron density away).
Like with chemical hydrolysis, this is the SLOW step, the attack of the nucleophile.
This forms a tetrahedral intermediate, analogous to the chemical hydrolysis reactions we learnt about previously.
The carbonyl is then reformed, and the C-N bond breaks; the ‘extra’ $NH_2$ is ‘picked up’ from the same glutamic acid, regenerating the carboxylate (catalytic! The enzyme ‘active site’ is regenerated and ready to go again).
The C-terminal amino acid is now cleaved from the rest of the peptide.
Both diffuse out of the enzyme active site.
The zinc ion, the glutamic acid carboxylate, and another water molecule are ready to do the reaction again on another peptide.
The enzyme takes minutes to effect the hydrolysis, whereas acid (or base) hydrolysis in a flask/as a chemical reaction takes hours!

Angiotensin-converting enzyme (ACE) is part of the renin-angiotensin system.
ACE breaks down angiotensin I (a decapeptide) into angiotensin II (octapeptide) by hydrolyzing the second amide bond in from the C-terminus.

ACE-catalyzed peptide hydrolysis is a very similar mechanism to carboxypeptidase A.
An amino acid side chain reacts with the water that acts as a nucleophile and attacks the carbon of the $C=O$ amide.
Attack of the amide $C=O$ is possible because the zinc ion is coordinated to the $C=O$ oxygen.
… the next steps are also the same as before.

We can rationally design enzyme inhibitors by understanding how enzymes bond to and cleave their peptide substrates.
Various ACE inhibitor drugs have been designed, with the key feature of having a functional group that bonds well to the important zinc ion inside the enzyme active site.
- This ACE inhibitor binds tightly in the active site of ACE (bonding of the sulfur to zinc ion; the drug also mimics natural hydrogen bonding and ionic bonding) but does not undergo a reaction.
- The drug stays there, and the natural peptide substrate cannot bind to the enzyme, thus cannot be hydrolyzed – enzyme inhibitor.
ACE inhibitors are primarily prescribed to treat high blood pressure.