Enzymes II Flashcards
How can we tell an enzyme has a reaction rate limited by diffusion using glycerol?
- Measure the rate at which E + S makes E + P
- Alter the viscosity - Add glycerol to mixture, viscosity increases, diffusion is reduced. Does a change in viscosity (therefore velocity) slow the reaction rate?
- If so, the enzyme is diffusion limited and catalytically perfect.
e.g. Carbonic anhydrase
How can we tell an enzyme has a reaction rate limited by diffusion using theoretical calculations?
- Divide Kcat / Km (Or use the diffusion-controlled rate of encounter, 10^8 M-1S-1)
- Is the enzyme reaction rate for E + S -> E + P close to the diffusion-controlled rate of encounter?
How can we identify a perfect enzyme using free energy?
- Determine the actual free energy profile of the enzyme reaction. Are the diffusion steps rate limiting?
Describe how triosephosphate isomerase is a perfect enzyme.
- Catalyses conversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (3 carbon intermediates in glycolysis)
- Makes it so that instead of there being a big energy change and conversion of A to Z, it happens via intermediates that don’t have a high energy change between them, easing the conversion.
- Reaction is limited by E + S (substrate diffusion into enzyme), so decreasing energy levels doesn’t increase rate of reaction.
What do all proteases do, and name some examples
Hydrolyse peptide bonds
Serine, cysteine, aspartyl, metallo-proteinases
What two serine proteases are secreted by the pancreas and used in digestion
Chymotrypsin, trypsin
What do the 3 serine proteases (chymotrypsin, trypsin and elastase) have in common
They have a very reactive serine at their active site
When will chymotrypsin cleave/hydrolyse the peptide bond?
If the residue of the terminal is hydrophobic (Phe, Trp, Tyr)
When will trypsin cleave/hydrolyse the peptide bond?
If the residue of the terminal is positively charged (Lys, Arg)
When will elastase cleave/hydrolyse the peptide bond?
If the residue of the terminal is narrow/small
Why does chymotrypsin hydrolyse the bond when the residue is hydrophobic
Chymotrypsin has a hydrophobic pocket which fits with the hydrophobic side chain
Why does trypsin do what it do
Trypsin binding pocket has an Asp molecule which has a negative charge, producing an electrostatic interaction with the residue
Why elastase do sequence selectivity
Binding pocket for residue in elastase is narrow, blocked off by some valine residues, so it fits best with small residues.
Why are peptide bonds resistant to hydrolysis
Very high free energy of activation for hydrolysis (without an enzyme)
Describe the hydrolysis of the peptide bond
- Water attacks carbon atom of ketone of amide group in polyp chain
- Lone pair of electrons of oxygen help form tetrahedron intermediate, giving the O bound to the carbon a negative charge.
- The intermediate collapses, breaking the peptide bond and releasing the carboxyl group and the amide.
Why are serine proteases very effective at hydrolysing peptide bonds
Different reaction pathway to uncatalysed reaction, using Ser-OH group in the active site of every serine protease.
Describe the first step of the mechanism of serine proteases hydrolysing peptide bonds
- Ser-OH attacks the carbon of the ketone of the peptide bond
- Again tetrahedron intermediate formed and collapses.
- An ester is instead formed, an acyl enzyme intermediate with an ester bond to the polypeptide chain (Acylation)
Describe the second step of the mechanism of serine proteases hydrolysing peptide bonds
- Water comes in and attacks the acyl enzyme intermediate, forming a tetrahedral intermediate
- The tetrahedral intermediate then collapses, to release the enzyme and restore to its original state
- Carboxyl group and amide group released.
Why is enzyme serine-OH more reactive than a water OH
Within the active site of the serine OH, the serine is actually forming a hydrogen bonded network with the side chains of two other residues (histadine and aspartate)
This network forms a charge relay system, which pulls the proton off the serine OH and onto
Why is enzyme serine-OH more reactive than a water OH
Within the active site of the serine OH, the serine is actually forming a hydrogen bonded network with the side chains of two other residues (histidine and aspartate)
This network forms a charge relay system, which pulls the proton off the serine OH and onto the imidazole group, then to the aspartate.
The proton is shifted away from the oxygen, making the oxygen very electronegative. Easier to carry out nucleophilic attack.
What does the mitochondrial ATP synthase do
Synthesises ATP - the last step in oxidative phosphorylation. Driven by protons
Which subatomic particle is the inner mitochondrial membrane impermeable to
Protons (low on the inside, high on the outside, forming gradient)
Name the features of the enzyme rotary ATP synthase
Lollipop shape
Stator with 6 sub units, hole down the middle held in place by proteins
Rotating spindle in hole
Spindle in contact with 3 active sites, where ATP is made
Protons rotate rotor, registers the active sites to make ATP.
What happens when the rotor of the ATP synthase rotates
- ADP and phosphate are bound to an active site
- The rotor rotates 120 degrees
- Provides energy to release previously synthesised ATP in another active site of the enzyme
- This squeezes the ADP and the phosphate, to make ATP at the other active site
- This continues in a loop.
