Energetics and dynamics of protein action Flashcards
What is cleaving?
Removing the functional group or splitting metabolites into chunks.
What are hydrolases?
Hydrolytic cleavages
Nucleases - cleave nucleic acids
Proteases - cleave proteins
What are polymerases?
Polymerisation reactions - expanding RNA or DNA.
What are synthases?
Catalyses synthetic processes
What are kinases?
Important for signalling by phosphatases.
Adding or removing phosphate changes its interactions with other things.
What are isomerases?
Intermolecular rearrangment into other isomers.
What are oxido-reductases?
Oxidise or reduce substrates - dehydrogenases or oxidases
What are ATPases?
Use ATP to drive across membranes through motors and pumps.
What is the effect of high temperature on enzymes?
Heating an enzyme makes the molecules vibrate so it is harder to hold the substrate in place and collide and for the enzyme-substrate complex to take place.
At a high enough temperature the bonds will denature and structure degrade.
What does the graph for temperature and enzyme action look like?
Vo on y axis
Temperature on x axis
Gradual increase, sharp decline after peak.
What is the effect of low temperature on enzyme activity?
When cold the substrate and enzyme are moving slowly so are in the correct orientation less often and won’t bind as often so low rate.
The side chains move slower to accomodate substrate.
What is the effect of pH on enzyme activity?
The charges on amino acids change so the structure changes and the substrate binds less easily.
At extreme pH the protein is denatured.
What does the graph of pH on enzyme activity look like?
Vo on y axis
pH on x axis
2 lines - first peak is narrow at low pH.
broad peak at high pH
What is the progress of a reaction?
Substrate + enzyme –> enzyme substrate complex –> enzyme product complex –> release enzyme + product
What does the free energy of a reaction look like?
Free energy on y axis
Progress of reaction on x axis
Activation energy peak
Free energy of enzyme and product is lower than enzyme and substrate at start
What does the free energy look like when an enzyme is added?
Peak at activation energy is lower
Enzyme and product energy is at same point as original line.
How does an enzyme affect the free energy in a reaction?
Lowers the activation energy so it is easier to form the transition state.
What is an exergonic reaction?
Spontaneous - ΔG is negative and energy is given out.
What is an endergonic reaction?
Not spontaneous
Energy is taken in and ΔG is positive.
What is a freely reversible reaction?
When ΔG = 0 it is freely reversible.
see graph
What is an energetically unfavourable reaction?
Endergonic because ΔG is positive
There is a transition state, with a peak which needs overcoming.
An enzyme will lower the peak of the activation energy to the transition state but still requires energy.
See graph
What is an example of coupling exergonic and endergonic reaction?
Phosphorylation of glucose to produce glucose-6 phosphate.
Large ΔG so not spontaneous so couple with a spontaneous reaction.
Add ΔG together to form an overall negative ΔG so the reaction will be favourable.
What is the induced fit model?
Proposes distortion of enzyme and substrate to favour product formation.
Active site has similar structure to substrate, forms transition state, substrate binds to enzyme and substrate takes a form so it is easier to form products.
What is catalysis dependent on?
Localisation, orientation and binding energy of substrate.
Catalytic residues on protein framework.
These all contribute to a lower energy of the transition state.
Why is the enzyme complementary to the transition state?
Because this lowers the activation energy.
How does hexokinase show enzyme function?
When there is nothing bound, hexokinase has an open structure.
When glucose and then ATP bind, it forms a closed structure around it, which brings the catalytic residues closer together so the reaction can happen.
How does enzyme substrate binding affect water?
There are spheres of water around the enzyme and substrate joined by hydrogen bonding.
When the substrate binds to enzyme, the water molecules move and hydrogen bonds break, so water is more disordered.
This increases entropy and ΔG decreases, so enzyme substrate complex has lower free energy.
What is the basic reaction rate of an enzyme driven reaction?
Initial: substrate is abundant so easy to bind
Curve levels off as substrate is used up until it is very limited because there is so little substrate that it cannot form enzyme substrate complexes.
What is the Michaelis Menten equation for initial rate?
E + S <–K-1 –K1–> ES –K2–> EP
K1 is rate in forwards reaction.
K-1 is rate in backwards reaction
What is K2?
K2 = Kcat which is the turnover number
It is the speed of the reaction and can compare between enzymes.
What is the slowest rate of reaction?
The slowest step determines the overall rate of reaction.
K2/Kcat is the slowest rate.
The rate of E+S is so fast it isn’t measured.
What is the steady state assumption?
[ES] remains the same.
As soon as ES forms EP, another molecule of ES is formed.
What are the assumptions of the M-M scheme?
The reverse reaction is neglible/insignificant.
Only a single central complex (ES) forms - no EP forms.
More [S] than [E].
The interaction of S with E to form ES does not significantly affect [S].
What is initial rate?
V0
V for velocity is on vertical axis
[S] is on x axis
What is Vmax?
The curve goes as high as possible.
The maximum velocity is Vmax but the curve never reaches it.
The enzyme is fully occupied by substrate.
How is Vmax calculated?
Vmax = Kcat[E]t
[E]t is the total amount of enzyme.
What is KM?
The Michaelis constant
Km is the substrate concentration where the reaction rate is half maximal.
What is the Michaelis Menten equation?
Vo = Vmax[S] / Km + [S]
What is the rearranged Michaelis Menten equation?
Vo = (Kcat[E]t)[S] / Km + [S]
What is the equation linking Kcat and Km?
Kcat/ Km is a measure of enzyme efficiency - for comparing enzymes.
A large Kcat (rapid turnover) or small Km (high substrate affinity) gives a large number
What is catalytic perfection?
3x10^8
Carbonic anhydrase has catalytic perfection.
What are the limitations of the Michaelis Menten scheme?
Does not explain kinetic properties of many enzymes.
Does not take into account secondary substrate binding affinity.
Does not work for allosteric enzymes - produces sigmoidal curve - can estimate Vmax but not Km.
What are the units for Kcat?
Per time unit, so can instantly discount some answers in mcqs.
What are the units of rate of reaction?
Change in concentration per time.
What are the units for Vo?
Vo is rate so Concentration per time
e.g. micromole per litre per second.
What is the Lineweaver-Burk plot?
Inverse of Michaelis Menten equation
1/Vo = Km/Vmax[S] + 1/Vmax
see picture
What is the equation of a straight line?
y = mx+c
y = y axis
m = gradient
c = y intercept
How does the Lineweaver Burk plot fit the equation of a straight line
1/Vo = y
Km / Vmax = m
1/[S] = x
1/Vmax = c
What value of Lineweaver Burk is the x intercept?
The x-intercept is -1/Km
What is the limitation of Lineweaver Burk plots?
It is highly sensitive to measurements at low substrate concentrations [S].
-1/Km is greatly affected.
So should do lots of data points at low [S].
What is competitive inhibition?
Inhibitor is the complementary shape of the active site of enzyme and blocks substrate from binding and undergoing catalysis.
What happens when you increase inhibitor concentration in competitive inhibition?
Less chance of substrate binding to enzyme, until it reaches a point where no substrate binds.
Vmax is unaltered.
Km increased because inhibitor is in the way so substrate doesn’t bind - affinity seems less.
What is non-competitive inhibition?
Inhibitor is different shape to substrate and binds to enzyme at site other than active site.
It modifies enzyme conformation - quaternary structure - so can’t form product as fast or release product.
Inhibitor has high affinity for second binding site.
How are the Michaelis Menten values affected by non-competitive inhibition?
Km is unaltered (affinity for substrate)
Vmax reduced so rate less.
Kcat - how fast enzyme can work - decreased
What is the graph for competitive inhibition?
At half vmax the Km of no inhibitor is smaller than with inhibitor.
What is the graph for non-competitive inhibition?
Half Vmax - Km is unchanged from no inhibitor
What does the Lineweaver-Burk plot look like for competitive inhibition?
1/[S] on x axis means if [S] or Km gets bigger, the x number gets smaller and moves towards y axis.
What is the Lineweaver-Burk plot for non-competitive inhibition?
Y axis is 1/Vo so if Vmax is reduced then number gets bigger and moves up y axis.
What are examples of naturally occurring competitive inhibitors?
Digitalis - foxglove, Na+/K+ ATPase
Tetradotoxin - puffer fish, Na+ channel
What are examples of synthetic competitive inhibitors?
Ibuprofen - COX enzyme inhibitor, anti inflammatory
Sulfanilamide - diydropteroate synthetase, antibacterial
What are examples of naturally occuring non-competitive inhibitors?
Caffeine - cAMP phosphodiesterase, glucose transport
What are examples of synthetic non-competitive inhibitors?
Haloperidol - nitric oxide synthase inhibitor, anti-psychotic.
Trichostatin A - histone deacetylase, anti-cancer
What is non-competitive inhibition in biosynthesis of isoleucine?
Threonine is catalysed by threonine deaminase to produce isoleucine.
Isoleucine can non-competitively inhibit TD.
As the isoleucine product increases, the first reaction rate decreases.
This leads to less isoleucine forming and a reduction in inhibition as the isoleucine dissociates from the enzyme.
Allosteric reaction.
What is neuraminidase?
Essential for viral proliferation.
Silidase - cleaves sialic acid.
What is sialic acid?
It is held in cleft mainly through its glycerol and carboxylate groups which form bonds with residues in the active site.
Positive charged Zanamivir (competitive inhibitor) adds other bonds, forming strong attachments to negatively charged sidechains at the bottom of the cleft. it binds better than natural substrate.
What is vascular adhesion protein-1?
In livers of alcoholics, there is lots of this enzyme which associates with scars formed by drinking.
Inhibitors which can block this enzyme activity could cure liver disease.
