Enzymes II Flashcards
What are perfect enzymes and what evidence supports this?
Perfect enzymes- reaction rates limited by diffusion
The perfect enzyme is the one where the reaction is limited solely by the binding rate, not the chemistry rate
Evidence?
1. Is the reaction rate affected by viscosity? Does a change in viscosity by adding glycerol slow the enzyme reaction rate?
2. Theoretical calculation for diffusion-limited reaction
K3/Km ~10^8 M^-1s^-1?
Carbonic anhydrase: k3 is ~600,000 s^-1; Km is 8 x 10^-3
3. Determine complete free energy profile for the enzyme reaction.
Is substrate-binding rate limiting
How do you calculate turnover number?
Turnover number (kcat/k3) is Vmax/[enz]total
What is an example of a perfect enzyme?
Triosephosphate isomerase is an example of a perfect enzyme and its part of glycolysis
Probably due to the intense evolutionary pressure that the process is as efficient as possible
It interconverts the dihydroxyacetone phosphate to glyceraldehyde 3-phosphate so that it’s not wasted
What are some characteristics of proteases?
Well understood enzymes
Therapeutic targets
Serine-, cysteine, aspartyl and metallo-proteinases (active sites) all hydrolyse peptide binds
What are serine proteases?
Serine proteases have a very reactive serine- attacks the peptide bond to form an acyl-enzyme
One of the serines can react with the peptide bond to form an acyl-enzyme intermediate which can then be hydrolysed very easily
Serine proteases hydrolyse protein peptide bonds but with sequence specificity
How do serine proteases show specificity?
These different serine proteases have a sequence specificity which is determined by the nature of the residue which is just on the N-terminal side of the peptide bond that will be cleaved
Chymotrypsin will only cleave peptide bonds where the determining residue is either phenylalanine, tryptophan or a tyrosine- all aromatic residues because it has a hydrophobic pocket
Elastase will only cleave a peptide bond is the determining chain is rather small because it has a narrow binding pocket
The binding of the serine proteases to specific peptide bonds is determined by their binding pocket
How do serine proteases catalyse the hydrolysis of a peptide bond?
Uncatalysed hydrolysis of a peptide bond:
Water attacks the carbonyl group of the peptide bond
OH attaches on to the carbon and we get what’s called the tetrahedral intermediate
The peptide bond is broken and the nitrogen picks up a hydrogen
Very high free energy of activation
Hydrolysis of a peptide bond by a serine protease:
Step 1 Acylation-Serine replaces water here and its OH attaches to carbon to form a tetrahedral intermediate and the NH2 is released to form an acyl-enzyme intermediate
Step 2 Hydrolysis of the acyl-enzyme intermediate- Much easier to hydrolyse an acyl-enzyme compound than to hydrolyse the peptide bond itself
Why is the serine OH much more reactive than the water one?
The serine OH interacts with two other residues in the active site- a histidine and an aspartate residue
These form a catalytic triad
There is a hydrogen-bonding network between the side chains of the histidine and the aspartate with the serine
The proton on the serine OH can be shifted onto the imidazole group
Called a charged relay cause you’re moving a proton from the oxygen molecule
The result is that the OH is much more negatively charged making it into a much more powerful nucleophile
A charge-relay system activates the catalytic serine by proton withdrawal
How is ATP synthesised in the mitochondria?
ATP synthesis in mitochondrion is by a proton-driven rotary ATP synthase
The inner mitochondrial membrane is highly impermeable to protons but during respiration protons are pumped from the inside of the mitochondria to the outside
So what happens is that you are storing charge and the concentration of protons between the outside and inside of the membrane is different
This is sometimes called a proton motive force
So you have an excess of protons on the outside compared to the inside
This difference is an energy source which can be used by the rotary ATPase which are plugged into the inner mitochondrial membrane
They can use the protons to actually synthesise ATP
How is rotary ATP synthase structured?
Rotary ATP synthase has 3 active sites activated by a rotating spindle
It is made of two parts
It has a rotor sitting in the membrane which can turn as a result of interacting with the protons
The rotor is connected to a spindle and that spindle protrudes through a hole in the middle of a complex
It is held in place by a protein link to the membrane
As the rotor moves around it causes the synthesis of ATP from ADP and phosphate and then for water to be eliminated between them with the synthesis of ATP
How can the rotation of ATP synthase be visualised?
U-tube
Engineered the ATP synthase to bind to actin which can be stained so that they could visualise the movement of the ATP synthase
How does topoisomerase II function to untangle the DNA?
It uses ATP to be able to bind a DNA segment from one chromosome and to pass it through a double stranded break that it introduces in the second chromosomes
And so by crossing one DNA duplex through another, this untangles the chromosomes and allows their ready segregation
Topoisomerase II and other type II enzymes cross a DNA segment from one chromosome through a transient dsDNA break in another
Topo II is a molecular clamp that unlinks tangled chromosomes
