Lecture 10 - Mechanisms for enzyme catalysis Flashcards
Covalent catalysis
Involves the formation of a reactive, short-lived intermediate, which is covalently attached to the enzyme
Nucleophilic site chain on the enzyme can include carboxylates, amino, S-, benzene-O-, CH2-OH (Ser,Thr)
What happens is that half of the substrate will bind to the enzyme. As a catalyst, this enzyme needs to be turned over again. Need to regenerate the active form of the enzyme which is done in a second reaction that is often done by using water as a second substrate (hydrolysis reaction)
Give an example of the type of amino acid side chain chemical functionality that is involved in covalent catalysis?
Sharing proton with histidine makes serinehydroxyl a good nucleophile.
What type of amino acids are likely to be involved in acid-base catalysis?
General base catalysis can be performed by the unprotonated forms of the amino acids listed above: Asp, Glu, His, Tyr, Cys, Lys. It is possible for a single amino acid residue to participate in both general acid and general base catalysis at different points in a single enzyme cycle
Acid base catalysis
Involves H+ transfer
Often involves charged amino acids such as Glu, Lys
His (pka approx 6.5) can donate or accept a proton
Enzyme activity is pH dependent
Each enzyme has a characteristic optimal pH at which its rate is highest.
Amino acid side chains need to be in the correct ionisation state for catalytic mechanism to proceed
Histidine in acid-base catalysis
Histidine is particularly suitable to these types of reactions.
Imidazole pKa ~ 6.5 – close to physiological pH
Depending on the environment of the active site His can donate or accept a proton
Chymotrypsin cleaves proteins
Proteases are hydrolyases. Substrates are a polypeptide and water. Products are two shorter peptides, or individual amino acids.
Chymotrypsin acts in digestion.
Other proteases make hormones, remove defunct proteins, or activate enzymes (as in blood clotting).
Divergent evolution of serine proteases
Same structure, unique specificities. Start with a common ancestor and evolve to get somewhat different functions and so can trace back. Difference between these is where in the sequence these cleave (chymotrypsin, elastase and trypsin)
Convergent evolution of serine proteases
Two distinct ancestors that have come together to find the same solution to some kind of physiological challenge. Same catalytic triad occurs in different order and in different structures. If they shared an ancestor, they should share order and structure however they do not. Cannot line up their primary sequences.
Chymotrypsin explained
Catalytic triad comprises serine, histidine and
aspartic acid. Sharing proton with histidine makes serine hydroxyl a good nucleophile to attack ‘scissile’ bond (a bond that is going to be cut)
His57, polarised by Asp102, withdraws a proton from Ser195, which becomes nucleophilic. (serine must be a good nucleophile because this is a nucleophilic attack, taking off a proton helps it become a better nucleophile and therefore it becomes more electron rich) (tkes proton off serine and puts it onto histidine)
Nucleophilic attack yields covalent, tetrahedral intermediate. Oxyanion hole stabilises the tetrahedral intermediate (as well as the negatively charged oxygen), lowering activation energy.
Non-polar environment of Asp102 raises its pKa.
Oxyanion-hole stabilises tetrahedral intermediate.
Tetrahedral intermediate decomposes, driven by general acid catalysis from His57.
Half of the polypeptide remains covalently attached to the enzyme in an ‘acyl-enzyme intermediate’. The other half can leave the active site.
Scissile bond is now broken because the N has now made a new bond up to the proton that it got from the histidine residue. Leaves us with a covalent intermediate with the other half of the polypeptide we are breaking still bound to the serine. We now have to regenerate the enzyme by moving the reaction in some way.
Water replaces one of the products in the
active site. Replace the product that is now complete with a nucleophile that is similar, so you drop a water molecule in where the N was and it has protons in similar places so can bind to the active site in a similar way and effectively it can now reverse the reaction we just did.
Polarised by His57 as a general base, water makes a nucleophilic attack forming a second tetrahedral intermediate, again stabilised by the oxyanion hole
With His57 acting as general acid, this intermediate
decays to form a carboxylate. The second product can leave the active site. The active site has been regenerated and is ready for another round of catalysis.
Chymotrypisin only cuts peptide bonds next to aromatic amino acids, able to position the bond next to important side chains for subsequent attack
Purpose of the catalytic triad
Purpose is to pull the proton off the serine and make it a better nucleophile so it can attack the carbonyl carbon that is the electqrophil in the nucleophilic attack
Catalytic triad set up
- aspartic acid = polarises His by attacking the proton (higher aka due to hydrophobic pocket and therefore doesn’t want to be charged)
- histidine = grabs the proton from the OH of serine
- serine = OH to O- so now it is a good nucleophile
Summary
- aspartic acid is a primer of His (activates His)
- His undergoes acid-base catalysis
- Serine is a nucleophile
Peptide bond cleavage involves BOTH
Acid base catalysis and covalent bond catalysis
Aspartic protease mechanism
2 active site residues are the 2 Asp.
Carbonyl carbon is a decent electrophile therefore water can attack and act as a nucleophile.
Asp (LHS) is using acid-base catalysis in early steps, Asp (RHS) uses acid-base catalysis in later steps. 2 Asp close to each other so it is not likely that they are going to be negatively charged at the same time, there is going to be a strong repulsion so charging one of them is easy but charging both of them is likely to be more difficult since they will be same charges (deprotonation of the first one influences the deprotonation of the second one)
HIV protease and pepsin are examples
Best to worst nucleophile
Hydroxide
Hydroxyl - the uncharged side chain of serine
NH3+ - the positively charged side chain of lysine (needs a lone pair to become a better nucleophile)
Methyl - a methyl group on the side chain of valine (never ionisable)
