enzymes II Flashcards
What is a perfect enzyme?
Perfect’ enzymes have reaction rates limited by diffusion .
evolution has improved the chemistry of the 2nd step such that the rate limiting step if the diffusion of the enzyme and substrate together
in an enzyme substrate reaction, which part can evolution work on?
, a natural process by which E and S diffuse together in solution to form the ES complex.
The second step is to do with chemistry. Evolution can work on this step, by mutations it can improve the properties of the active site so that the free energy of activation for this step is reduced, therefore evolution can improve the chemistry of the reaction.
- the overall reaction rate is determined by the slowest step.
Evolution cannot improve the physical nature of the first step, ie diffusion, as its determined by the properties of the enzyme and substrate.
In this process, the overall reaction rate is determined by the slowest step. By improving the chemistry, we will reach a point where the limiting step Is the diffusion step.
outline 3 experiments that can determine whether an enzyme is perfect and that diffusion is the rate limiting step.
If diffusion is the rate limiting step, then there overall should be a reduction in the rate of product formation.
- Altering the viscosity of the solution. Is the reaction rate affected by viscosity?
Add glycerol to the reaction mixture- it makes the solution more viscous and slows-down the rate at which E and S diffuse together to form ES. If a change in viscosity slows the enzyme reaction rate then the enzyme is diffusion limited and so perfect. eg: carbonic anhydrase. - A second approach to identifying whether an enzyme is perfect and limited by diffusion is by theoretical calculations.
. if the enzyme is diffusion controlled, then the rate constant should be 108.
Carbonic anhydrase: k3 is ~600,000 s-1 ; KM is 8 x 10-3M - free energy profile. If the diffusion steps are rate limiting then the enzyme is perfect.
- A lot of work to obtain free energies for all the reaction species and transition states but yields hard data that enables you to see whether the diffusion steps are limiting overall for the enzyme
e. y triosephosphate isomerase
why is triosephosphate a good catalyst
- The role is to triosephosphate is to interconvert dihydroxyacetone phosphate to glyceraldehyde 3-phosphate.
- The result of this Is that both C3 halves of glucose can be utilized to make ATP.
- During evolution, triosephosphate isomerase would be under intense evolutionary pressure to be a good catalyst as having a functioning and efficient glycolytic pathway would be crucial for survival because it makes ATP needed for muscle contraction
how can we tell that triosephosphate is a perfect enzyme?
the free energy profile
- The enzyme and substrate forms the ES complex – diffusion step.
- The ES complex is converted into an enzyme intermediate complex EZ – chemical step
- EZ is converted into the enzyme bound product – chemical step.
- The product is released – diffusion step.
- In the free energy the rate limiting steps are the diffusion steps because evolution has reduced the chemical steps to the point where they are less efficient than the diffusion steps.
- There is no point in the chemistry reducing the free energies of activation of these steps any further because it doesn’t affect the overall rate of the reaction.
serine proteases are an example of?
reducing the free energy of activation by offering a different reaction pathway
describe serine proteases and how they react
- Hydrolyse peptide bonds
- The enzymes take place in the chemical steps involved in the hydrolysis of the peptide bonds . it forms an acyl enzyme intermediate with the substrate.
]- 3 serine proteases: Chymotrypsin and trypsin, both secreted by the pancreas and involved in the digestion and elastase - All three of these enzymes, and other serine proteases have an active serine at their active sites that attacks the peptide bond to form an acyl enzyme
describe the structure of chymotrypsin
serine-195 is present in a groove that binds the polypeptide chain that is going to be hydrolysed
- The active serine-195 is present in the binding pocket of the active site cleft . it participates directly in the catalytic reaction. It takes part in the chemistry of the hydrolysis of the peptide bond.
- It is close to two other residues, histidine-57 and aspartly-102. The hydrogen bonded network between the sidechain of these residues are responsible for potentiating the reactivity of serine. catalytic triad – feature of all serine proteases.
how to serine proteases hydrolyse peptide bonds?
Serine proteases hydrolyses protein peptide bonds with sequence specificity. They don’t hydrolyse every peptide bond
why do serine proteases hydrolyse with sequence specificity?
it depends on the nature of the residue on the n terminal side of the peptide bond that’s going to be hydrolysed. Chymotrypsin prefers to cleave this bond when the residue is hydrophobic (ph,try,tyro)
- Typsin cleaves the bond when the residue is positively charged, lysine or arginine
- Elastase cleaves the bond when the residue is small .
- X ray crystallography explains why :
- Chymotrypsin has a hydrophobic pocket with asp at the bottom which accommodates the residues which are also hydrophobic. The binding pocket of trypsin has a negative charge which can form an electrostatic attraction when the residues are positive. In elastase, the binding pocket is narrow due to the valine residues
- They show selectivity due to the nature of the pockets which bind to the reside.
why are serine proteases effective at hydrolysing peptide bonds ?
- They use a different reaction pathway to hydrolyse the bond compared to the uncatalyzed reaction
- To do this they use the highly reactive serine-OH group in the active site of all serine proteases, which catalyses the breakage of the peptide bond by a 2 step mechanism.
- Step 1, acylation: Instead of attacking with water (uncatalyzed reaction) uses serine-OH and attacks the carbonyl group of the peptide bond.
- Forms the tetrahedral intermediate which collapses. An acyl enzyme intermediate which has an ester bond forms. The amino terminal end of the pb is released.
- Step 2 : water attacks the acyl enzyme intermediate. The water attacks the ketone group to form a tetrahedral intermediate which collapses. This releases the enzyme which returns to its original state. In the process, it releases the carboxyl terminal end of the cleaved peptide bond.
- The attack of water on the acyl enzyme intermediate is easier than the attack of water on an amide group, thus this different reaction pathway hydrolyses the peptide bond using 2 steps with lower free energies of activation than that of the uncatalyzed reaction.
why is serine OH more reactive than OH
In the active site, serine-oh forms a hydrogen bonded network with the side chains of histidine and aspartate. This hydrogen bonded network forms a charge relay system which tends to pull the proton off serine-OH and onto the imidazole group and then the proton on the imidazole group is shifted onto the aspartate. the proton is being shifted away from the oxygen of serine which makes the oxygen very electronegative, therefore its able to carry out its nucleophilic attack on the peptide bond.
- The charge relay system formed by the catalytic triad of amino acids causes the effective attack of the Serine-Oh from the enzyme
what forms the catalytic triad?
serine-195, histidine-57, aspartyl-102
what is conserved among members of serine proteases ?
the 3D structure and the cleft for protein hydrolysis is conserved among members of the serine proteases.
-charge relay system
serine proteases hydrolyse peptide bonds through which intermediate ?
acyl-enzyme intermediate
