Biochemical Basics - Week 2 Flashcards
What is the active catalytic site of an enzyme?
-The region where the reaction occurs.
-Not always the same site as where the substrate binds. Can have a range of
functional groups present including coenzymes, metal ions and amino acid residues.
What is a transition site of an enzyme?
In order for the reaction to occur, the substrate needs to be activated by functional groups, and during the reaction a temporary high energy intermediate, known as the transition state will be formed.
Why don’t enzymes alter the position of
reaction equilibrium?
- These complexes are in equilibrium.
- They can form and they can fall apart.
- The enzymes don’t invent new reactions and so don’t affect the position of equilibrium, or the balance of the rate of formation and breakdown of the substrate in the product.
- They just allow it to occur quicker, so you reach a point of balance faster.
What is the ‘lock and key’ model?
-The idea that a substrate must recognise the substrate-binding site with its aptitude and that it must be identical, or at least complementary in order to successfully bind.
-By complementary, it means the residues are going to display the counterparts to residues or functional groups found within the substrate binding site.
-So, for example, if there are
hydrogen donors in the binding site on the enzyme, there has to be hydrogen acceptors direcetly opposite on the
substrate, or negatively charged residues, which will complement the positively charged residues directly opposite them in the substrate binding site.
-So there is a geometrical complementarity that must be met in order to permit the
substrate to bind.
-Substrate will bind through hydrophobic, electrostatic interactions and hydrogen bonds.
-Binding can be prevented by steric hindrance and charge repulsion.
What is the ‘induced-fit’ model of enzymes?
- As substrate binds, enzymes undergo a conformational change, so the enzyme is going to change slightly in order to bind it better.
- So it has a dynamic surface.
- Side chains of amino acids (active site) reposition and binding interactions increase.
How is the active site of an enzyme formed?
-Formed by a cleft or crevice within the polypeptide chain, or it’s formed by two polypeptide chains, so two subunits, forming your catalytic site.
-That’s the benefit of having a multi-subunit protein.
-A range of functional groups provided by the side chains of the amino acids that make up the catalytic site, will permit the catalysis to occur.
-It could be cofactors or coenzymes.
-The substrates will be so arranged in 3D space that they will approach each other, or they will be arranged with the correct geometric orientation such that these functional groups can then interact with the substrates and permit catalysis.
-As the substrate binds, may find an enhanced interaction is permitted by the functional groups within the catalytic site. -They’ll start to form temporary bonds with the substrate, and as that changes into the product, it will go through a high energy intermediate called the transition site and it will stabilise this
intermediate.
-Once the transition state is passed, and the product is being produced, any temporary bonds that have
been made to stabilise the transition site are going to fall away.
-That will permit the prodcuts to merge, and then the
enzyme returns to its original form and is free to bind to other substrates.
What is the transition state complex of an enzyme?
-The higher energy intermediate between the substrate and product.
-It can be the point where bonds are maximally
strained or the point where electron clouds are most significantly altered.
-If you were to look at a reaction profile and an uncatalysed reaction, you can plot the free energy available within the reaction or required by the reaction as the reaction progresses.
-Start at a certain level of free energy in your reactants which will climb to the maxima, the
amount of free energy required to create the transition state.
-Then it will drop to the minimum where the products are.
-That requires a significant amount of energy to achieve.
-If think about in terms of molecular theory and populations of molecules, you’ll realise that when you look at your population of molecules and the relative energy contained within,
be that thermal or kinetic, you’ll notice that originally in the underlying catalysed reaction, only a certain number of molecules will have the required energy to get all the way through to make the transition state successfully.
-So only have a small number that will reach that level.
-With stabilisation permitted by the catalytic enzyme, the amount of energy required to form that higher energy
intermediate is less.
-That means the number of molecules with the required energy gets higher.
-The proportion of molecules that have that lower energy level is bigger so that means more molecules are capable of going through the reaction which means the reaction rate occurs quicker.
-So the overall rate of reaction is determined by the number
of molecules acquiring activation energy and enzymes decrease the activation energy.
What is activation energy?
Differenence between substrate and transition-state complex.
What are cofactors and coenzymes?
- The catalytic properties of enzymes often depend on non-peptide molecules, called cofactors or coenzymes.
- Coenzymes tend to be organic molecules and cofactors can be metal ions.
- If a cofactor is tightly bound, can be known as a ‘prosthetic group’.
- Coenzymes are usually synthesised from vitamins, which can be fat or water soluble.
- If deficient in vitamins, won’t be able to make coenzymes, which can lead to disease.
What are two examples of water soluble
vitamins that make coenzymes?
-Thiamine is a vitamin used to make a coenzyme called thiamine pyrophosphate, which is involved in a range of
reactions, including reactions catalysed by pyruvate dehydrogenase.
-If don’t have it in diet, can develop a disease called
BeriBeri, which causes tachycardia, vomiting and convulsions.
-Nicotinic acid is a vitamin that is precursors of NAD+ and NADP+.
-If deficient can lead to Pellagra, causing dermatitis, diarrhoea, dementia and death.
Why is pH important in enzyme action?
- Active groups within the side chains of proteins have different abilities to dissociate or associate depending upon the surrounding pH.
- Their pKas, the measure of how well they can dissociate, varies depending on the amino acids, and as proteins can be made of many different amino acids, then the ability of a protein to retain its structure can vary depending on the pH.
- For example, pepsin, which occurs in the digestive tract, works at pHs that are acidic.
- If pH of surrounding solution moved away from optimum, could cause different amino acids to dissociate, and tertiary structure will be lost.
Why is temperature important in enzyme action?
-Human enzymes function optimally at 37 degrees Celsius.
-Increasing temperature from zero to 37 degrees increases
reaction rate and there is greater vibrational energy of substrates.
-Proteins have a capability to withstand thermal energy, but if it gets to great, the structure will become too rigid to do its part, so won’t have flexibility required.
-Higher temperatures lead to denaturation, meaning loss of secondary and tertiary structure, and loss of reaction rate
What are isoenzymes/ isozymes?
-Enzymes that catalyse the same reaction, but have a different structure to one another, so have different Km and Vmax.
-The enzymes differ in amino acid sequence.
-Lactate dehydrogenase is an example.
-It’s a tetramer from two
subunits, and it can be formed from a range of monomers, so can actually lead to five different versions of enzyme,
which are going to be structurally different to one another.
-LDH1 = H4 and is made of four monomers, which are the
heart form (H=heart).
-LDH5=M4 and is made of four monomers of the muscle form.
-LDH2=H3M, LDH3=H2M2 and
LDH4=HM3.
-Knowing this is useful as if you know which organs have certain isozymes, can detect when things start going wrong.
Why is diagnostic enzymology important?
-Can measure enzyme activity and concentration, especially in the serum, and that can give you certain types of diagnostic information.
-Enzymes in serum are divided into three categories.
-(1) Serum specific enzymes - enzymes that are meant to be there, so are in their normal location, e.g. enzymes involved in blood coagulation.
-(2) Secreted enzymes - enzymes that tend to be part of the circulatory system as they’ve been secreted for some reason, but are
going somewhere else to do a job.
-(3) Non-serum specific enzymes - no physiological role in serum, within the
circulatory system, but released due to cell turnover, damage, morphological changes, malignancy.
-They are the ones that shouldn’t be present.
-If your cell is damaged for a variety of reasons, it can release its contents over several hours and that will be facilitated by concentration gradients across your cell membrane.
-Cell membrane can be
damaged by reduced oxygen for example.
What are transition state analogues?
- As the transition state complex binds more tightly to the enzyme than the substrate, extra bonds can be transiently formed to stabilise them.
- Compounds that resemble its electronic and 3D structure, analogues of the transition state complex, can be more potent inhibitors of an enzyme than say something to designed to act like the substrate.
- The analogues get in the way of the active site, so could potentially be used as a drug, and would be very highly specific for the enzyme they are designed to inhibit.
- Unfortunately, however, the transition state is unstable, it will either become a product or will degrade back down to its substrate, especially if not bound to the enzyme, as the enzyme stabilises it.
- So it would be difficult to make a drug that resembled transition state analogue and have it survive getting through the digestive tract or it moving from the injection site to the site of action.
- So some approaches in drug design are trying to deal with the instability problem by designing drugs that are almost like transition state analogues, but then they have a stable modification added to them so they produce a stable prodrug that then becomes the stable transition site analogue at the site of action.
How can you use a transition state analogue to design a complementary antibody?
-If you have the structure of a transition state analogue modelled, it can be used as an antigen for the production of an antibody.
-It would mean that the antigen binding region of the antibody came to represent the catalytic site of the
enzyme.
-So a catalytic antibody is produced, known as the ‘abzyme’.
-So the arrangements of amino acids side chains
in the variable regions of the antibody end up have a similarity to the active site in the enzyme at the point at which the transition site is made.
-Consequently, you can make an artificial enzyme.
What is an equation of enzyme kinetics?
-E+S⇋[ES]→P+E
-First half of this reaction equation, can say formation of enzyme-substrate complex is reversible.
-It can form, but also break apart.
-Formation of products is an irreversible reaction, so
enzyme won’t bind product and make enzyme-substrate complex.
-When trying to categorise an enzyme reaction, need
to work out how quickly it does its job.
-So trying to determine set of mathematical quantities known as the rate constants, terms that allow you to define the speed and direction at which different parts of your reaction occur.
-Forward reaction going from enzyme plus substrate to enzyme-substrate complex is K1, and backwards reaction is
K-1.
-The second arrow, the irreversible part of the reaction, is K2 or Kcat.
What does a concentration-time graph of enzyme action show?
-Can use to understand enzyme function.
-Concentration of relative components on y axis and time on x axis.
-Plot varying concentrations.
-Then use rate constants to explain why graph look as does.
-Two separate sections on
diagram.
-Pre-ready state is the first few hundred milliseconds of a reaction and will need special equipment to measure
this.
-It’s a very short period of time where enzyme is with an excess of substrate and product starts to gradually build up.
-After that particular point, the reaction rate and concentration of your intermediate generally change fairly slowly over time.
-So the concentration of free enzyme has decreased to a more stable level, as has the enzyme substrate complex.
-So when trying to work out how an enzyme performs, work in steady state period of its reaction to find
rate constants.
What graph can you plot when you’ve challenged an enzyme with different substrate
concentrations?
-V0 is the initial rate against substrate concentration.
-So will have performed a range of experiments where challenged an enzyme of fixed concentration with a variety of different substrate concentrations.
-For each substrate concentration, plot a conc-time graph, and calculate the initial velocity for each - can then plot graph of V0 against substrate concentration.
-The initial rate will vary hyperbolically with substrate concentration.
-A hyperbolic
curve is an open ended curve - so a smooth open curve that’s tending towards an upper limit, Vmax.
-So will have an enzyme producing enzyme-substrate complex, which will increase as the substrate concentration increases, and the
enzyme-substrate complex can dissociate back to free enzyme and substrate.
-This behaviour can be explained by the Michaelis-Menten equation which is Vmax[S] all divided by Km+[S}.
What is the Michaelis menten equation?
-Relates the initial rate to a couple of quantities - substrate conc, [S], and Vmax and Km.
-Vmax is the maximum velocity of your enzyme, the fastest it will work for a set concentration.
-Km is the Michaelis Menten constant, the concentration
of enzyme where you reach half maximum velocity.
-Calculate these values as they are a good way of allowing a
comparison of enzymes at a specific concentration of enzymes.
-Michaelis Menten equation has a number of assumptions that underpin it.
-There’s the idea that the number of molecules in your substrate is much larger than the number of enzymes and that this means that the percentage of your enzyme bound substrate is actually low at any one time.
-Another assumption is that you’re not working with a multi-enzyme complex.
-If you were to plot initial rate against substrate concentration by multi-enzyme complexes, you’ll note they don’t have this kind of curve, but an S shape, suggesting the subunits are interacting with one another and affecting performance abilities of the other substrates.
How is the Michaelis Menten graph useful at understanding the behaviour of an enzyme?
-Two particular sections of graph are useful at showing this.
-Top rightmost section of graph - as increase substrate
concentration, initial rate don’t vary too much.
-This happens when your concentration of your substrates are much greater than the Km constant.
-This part of the graph is telling you you’ve got zero order kinetics or saturation kinetics occurring.
-There’s so much substrate present, that all of the active sites are occupied, and the reaction no longer depends on substrate concentration, it becomes independent of that.
-Can’t form any more enzyme-substrate complexes
as it’s being formed at its maximum rate, and so in which case the rate of reaction is only dependent on how quickly the product is released from the enzyme and then the enzyme is free again.
-Left side of graph, at bottom - displays first order kinetics, where the rate of reaction is proportional to substrate concentration.
-Such low substrate concentration here that the occupancy of the active sites on the enzymes are low, meaning the enzymes are desperate to encounter more substrate.
-As provide more substrate in various reactions, they can work faster, so the rate in the
early part of the graph is proportional to the substrate concentration.
What is Vmax?
-Reaction rate at which all the enzyme molecules contain bound substrate, so the amount of product being produced in a set period of time.
-If were to add more enzyme, amount of product can produce within that set
amount of time also increases. -So if vary amount of enzyme, Vmax will also change.
What is Km?
-Substrate concentration at which the initial rate is half the maximum velocity.
-Although velocities will change depending upon the amount of enzyme present, the concentration at which half of that velocity or maximal velocity is reached will not change.
-Look at this constant because enzyme velocity is most sensitive to change in substrate concentration just below your Km value.
-If you were to change substrate concentration at concentrations that were theoretically close to Vmax, you’d have to change them quite a bit.
-If working with substrate concentration in the vicinity of Km however, you don’t have to increase them or decrease them very much in order to have a great swing in the rate of reaction.
-If you have low Km, means you’ve got an enzyme with high substrate affinity, so only need a little bit of substrate to be
present before half the active sites in your enzyme population are filled.
-If you’ve got a high Km enzyme, has a low substrate affinity, so need lots of substrate around before reach concentration where have half a maximal velocity.
How can you compare isozymes?
-Isozymes are enzymes that can catalyse the same reaction but have slightly different structures, so different kinetic
properties and therefore show different kinds of abilities.
-Example is isozymes of hexokinase, a type of enzyme often involved in the metabolism of glucose.
- It adds a phosphate from ATP to glucose forming glucose 6-phosphate.
-Once it does that, it locks it in position in the cell and is metabolised.
-Many cells, for example red blood cells, will use glycolysis to create ATP and hence drive their function.
-Others, for example liver cells, store glucose in a storage
polymer called glycogen.
-In order to have these varying abilities, have to employ different isozymes, which have
different kinetic abilities.
-If look at hexokinase I, the isozyme found in red blood cells, has a low Km, so its concentration when it reaches half maximum velocity is actually 0.05 millimolar, mM.
-Glucokinase though, found in liver, has a high Km, 5-6Mm.
-In red blood cells, want enzyme to fuel activity of red blood cell.
-Red blood cell doesn’t have mitochondria in mature form, so requires glycolysis to give it enough energy to function.
-If it doesn’t, membrane pumps won’t work and end up having ruptured red blood cell.
-Without red blood cells working, not moving haemoglobin around.
-So need enzyme to work at a decent rate at very low levels of glucose.
-So these isozymes have a high affinity for the substrate
of glucose, which means have a low Km.
-Glucokinase, however, has a different job, to metabolise glucose or start it on
the metabolic pathway to being stored as glycogen, which only want to do when glucose is in excess.
-Otherwise liver would take up all glucose, lock it in hepatocytes by adding the phosphate group and then it all gets stored as glycogen
and your red blood cells wouldn’t work.
-So only want liver to perform this function when lots of glucose about, which is
why glucokinase doesn’t do much until there’s a high enough amount of glucose, around 5-6Mm.
What are the different types of inhibitor?
-If an inhibitor is not covalently bound to an enzyme, it’s a reversible inhibitor as can remove inhibitor from enzyme by dialysis.
-Can characterise the behaviour of an inhibitor by measuring the inhibition constant, Ki, the concentration of inhibitor required to reduce the maximal velocity by half.
-Can also characterise inhibitors with respect to the relationship of the substrate normally used by the enzyme, so they could compete with the substrate - in this case they’d be competitive inhibitors.
-Also have non-competitive and uncompetitive.
-In most reactions, the products that are produced
can be considered reversible inhibitors of the enzyme, and are often considered competitive.
What are competitive inhibitors?
- Competitive inhibitor will compete with the substrate at the substrate recognition site.
- If were to measure initial reaction velocities at a range of substrate concentrations in the presence and lack of the competitive inhibitor, would see a hyperbolic curve.
What is the effect of competitive inhibitors on Vmax and Km?
-As inhibitor is binding to the same site as a substrate, find that can overcome effect of reversible inhibitor by increasing substrate concentration.
-So when substrate concentration is reached to a relatively high level, substrate binding sites are occupied solely by substrates and the inhibitor molecules can’t bind, which is why Vmax, the maximal velocity of
the enzyme, won’t actually be affected, it will remain the same.
-However, the concentration of substrate required to
reach half Vmax does change - can refer to as Km apparence, so the apparent Km in the presence of an enzyme - it
gets larger.
-This is because need to provide more substrate to occupy half the active sites within the enzyme population
in order to reach half of the maximal velocity, as they’re going to be competing with the inhibitor for presence in those sites.
-The apparent Km shifts or becomes greater, and Michaelis Menten’s constant becomes greater apparently in
the presence of the competitive inhibitor.
What is non-competitive inhibition?
-Dealing with a multi-substrate reaction, so need substrates A and B to be present in enzyme.
-Inhibitor binds in
substrate B’s recognition site, so it could be a competitive inhibitor with regards to substrate B.
-However, if thinking
about it in terms of substrate A, it’s actually considered a non-competitive inhibitor with regard to substrate A.
-So it doesn’t matter if change concentration of substrate A, that’s not going to prevent inhibitor from binding to substrate B’s binding site.
-Due to this, will see a lower Vmax.
-With respect to A, it’s going to lower the maximal velocity of the enzyme or lower the active amount of enzyme present within population, but with respect to A, Km won’t change.
