Biochemistry- Enzymes

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What 4 things to enzymes do?
1)Result in higher reaction rates 2)allow milder reaction conditions to be used 3)give greater specificity- no byproducts 4)give a capacity for control
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are all enzyme controlled reactions reversible?
Yes- those that are considered irreversible are only because the product will diffuse off (but if it were trapped then it would be reversible)
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Why can we consider only the forwards reaction when looking at enzyme kinetics?
because often when we measure enzyme activity we only follow it in the early stages of the reaction i.e. no product has been formed yet so cannot be reversed.
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What is activation energy?
an energy barrier (hill) that prevents reactants becoming products instantaneously. . An energy input is needed to convert reactants into unstable molecular forms called TRANSITION-STATE SPECIES
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Give two ways in which the energy barrier can be overcome
Heat- Speeds up all chemical reactions by raising the energy of the substrates closer to the barrier. 2)Pressure- reduces entropy in the system and hence increases the change of collision between molecules
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Why are neither of these particularly useful in biological systems? (2 reasons)
• our body does not have a wide temperature range that can be exploited… high temperatures denature proteins! • cells without defined cell walls are susceptible to damage by increased pressure.
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What do enzymes do to overcome these problems?
Provides an alternative pathway for the reaction to occur with a lower activation energy
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Why does the equilibrium constant remain unchanged?
both forward and backwards reactions are accelerated equally
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Look at the diagram on pg.2 of Biochemistry-enzymes1 notes. What does: a)ΔG‡uncat b)ΔG‡cat c)ΔΔG‡ represent?
a)Gibbs free energy of activation for the uncatalysed reaction b)ΔG‡cat is Gibbs free energy of activation for the enzyme-catalysed reaction c)the binding energy between the transition state and the enzyme
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Describe the 4 ways that an enzyme reduces the activation energy (first way)
1. an enzyme holds reactants (substrates) close together in the right orientation for the reaction, which reduces entropy and hence the reliance on random collisions UNLESS your enzyme acts so fast that it’s limited by the rate of substrate diffusion
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(next 2 ways)
2. an enzyme may put a “strain” on existing bonds, making them easier to break 3. an enzyme provides a “microenvironment” that is more chemically suited to the reaction
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4th way
4. sometimes the active site of the enzyme itself is directly involved in the reaction during the transition states (ie a different reaction path)
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Why is the active site of the enzyme not actually complementary to the substrate?
otherwise it would actually reduce the energy level of the substrate (making it more stable) creating an even greater energy barrier to break the stick.
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What are the two theories as to how an enzyme binds to the active site?
Lock and key model, and induced fit model
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Describe the lock and key model
the structure of the active site is complementary to its substrate like lock and key - this is now a rejected theory
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Describe the induced fit model
-flexible interaction between ligand and active site induces a conformational change -enzyme is in an open state and as it binds to substrate it is pushed towards transition state. - more widely accepted
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What else does this theory imply?
It implies that some enzymes will work on substrates they are not perfectly specific to (and hence can recognize multiple substrates), but that the reaction will be slower.
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In what two ways does the substrate tend to be specific to the active site?
Geometric- the active site is complementary to the structure of the substrate. Electronic- the amino acids in the active site interact specifically with the substrate
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What is Enantioselectivity?
the ability for an enzyme to react with one enantiomer and NOT the other → Can be use to discriminate between enantiomers
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What is stereospecificity a description of?
the reaction path, not the selectivity for substrates or products. Many enzymes are completely stereospecific i.e. the way the reaction occurs is exactly the same in 3D space.
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Different enzymes show a different degree of specificity. Some are specific to one substrate, whilst others aren't. Give an example of an enzymes that catalyses the reactions of a group of related molecules.
yeast ADH oxidises both primary and secondary alcohols, but ethanol is the most efficiently converted.
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Give an example of an enzyme which is very very permissive in specificity
digestive enzymes such as carboxypeptidase A can catalyse the hydrolysis of C-terminal peptide bonds to all residues except Lys and Arg and Pro
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Give an example of an enzyme which can catalyse different types of reacitons
chymotrypsin can catalyse peptide bond hydrolysis as well as ester bond hydrolysis
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What is the active site composed of? Will these residues necessarily be found together in primary sequence?
residues that come from different parts of the polypeptide chains. No, but they will come together and interact in 3D space when the protein is folded.
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What happened in 1961?
The first enzyme commission devised a system for classification and coding of enzymes
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What do the four numbers given to the enzyme encode?
1st number= one of 6 main divisions. 2nd number= subclass 3rd number= subsubclass 4th number= serial number
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What are the 6 different options for the 1st number to represent?
oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. (OTHLIL)
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What is an oxidoreductase? Give an example and what it does.
An enzyme which catalyses oxidation/reduction reactions e.g. alcohol dehydrogenase E.C. (aldehyde-->alcohol)
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What is a transferase? Give an example and what it does.
An enzyme that transfers functional groups e.g. hexokinase- (glucose +ATP --> G6P + ADP) E.C.
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What is a hydrolase? Give an example and what it does.
catalyse the hydrolytic cleavage of C-O, C-N, C-C and some other bonds, including phosphoric anhydride bonds carboxypeptidase A- EC (break down of polypeptide by hydrolyzing amide link)
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What is a lyase? Give an example and what it does.
enzymes cleaving C-C, C-O, C-N and other bonds by elimination, leaving double bonds or rings, or conversely adding groups to double bonds e.g. pyruvate decarboxylase- EC (pyruvate --> acetylaldehyde + Co2)
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What is an isomerase? Give an example and what it does.
catalyse geometric or structural changes within one molecule (isomerisation) e.g.maleate isomerase- EC (maleate -->fumarate)
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What is a ligase? Give an example and what it does.
catalyse the joining together of two molecules coupled with the hydrolysis of a diphosphate bond e.g. pyruvate carboxylase- EC6.4.1.1 (pyruvate + HCO3 + ATP --> oxaloacetate + ADP+ Pi)
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What are coenzymes?
Small organic molecules required by some enzymes during catalysis. They bind to the active site of the enzyme but are not considered to be substrates
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What are cofactors?
Same as coenzymes but are metal ions
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What are prosthetic groups?
Cofactors associated with the enzyme
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What are Cosubstrates
Coenzymes transiently (temporarily) associated with the enzyme
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What are holoenzymes?
A catalytically active enzyme-cofactor complex
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What is an apoenzyme?
Inactive protein (absence of the cofactor)
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Vitamins can act as enzyme precursors. Why is it therefore that they are found in an organism's diet?
. Many organisms are unable to synthesise parts of essential cofactors
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Vitamins in the human diet that are precursors are ...
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What kind of vitamins aren't components of coenzymes?
Lipid soluble vitamins (A and D)
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Give examples of 3 vitamins
thiamine pyrophosphate, coenzyme A, biotin (always bound)
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Give examples of 3 cofactors (sometimes bound, sometimes not)
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What do enzyme assays measure ?
either the disappearance of substrate over time or the appearance of product over time
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What are the three types of assay?
Fixed-time (discontinuous) / continuous / coupled
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Describe a fixed-time assay
The reaction is stopped (e.g. using acid or alkali) at set time points and the product/substrate is measured. This measures the initial rate of reaction before the product builds up.
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Why must A control reaction must be used whereby it isn’t incubated for any time?
just in case there is any background colour being formed.
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What is wrong with this method?
It is slightly tedious (as there are many flasks), and it can be difficult to measure the end-point
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What are the 4 possible units of an assay?
1)Activity/ Velocity- μmol/min (depends on amount of protein) 2)Specific activity - μmol/ 3)“Enzyme unit”- Amount which gives 1 mmol product/min 4)katal (SI)- Amount which gives 1Mol/sec
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Why is it much nicer to measure in specific activity instead of in katals?
katal's units are mols/sec therefore it is always going to be a very small number
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Describe a direct assay
Assay has a way of detecting product or substrate directly due to some property of either: –coloured product then colour change will be monitored and detected continuously using a spectrophotometer –Some substances absorb at a certain wavelength.
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Give an example
NADH absorbs light at 340nm –Fluorescent product –Gas product
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When would we used a coupled assay?
When neither product or substrate can be measured, but the product can be consumed in another reaction. The product of the next reaction can then be measured.
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Why must the enzyme catalyzing the second reaction be in excess?
so that the rate limiting step is the one to be measured.
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Give an example
e.g. the assay of a-glycerokinase (GK): Glycerol + ATP ----GK----> a-glycerol phosphate + NAD+ ----a-glycerol-phosphate dehydrogenase---->dihydroxy-acetone phosphate + NADH + H+. The production of NADH can then be measured.
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... see handwritten cards for Q's
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Why are enzymes usually only active in a narrow pH range? (4 reasons)
• pH sensitibity of substrate binding • reduced catalytic efficiency of the enzyme • ionization of substrate • protein structural changes (usually occurring at extreme pH’s)
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What can kinetic analysis as a function of pH provide?
information about the nature of functionally (catalytically) important residues.
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What does the general chemical equation for enzymes with two functional groups assume?
That only EH and ESH are active
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At each pH, what do the enzymes behave according to?
The simple Michaelis-Mentin equation
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What does pH therefore modulate the amount of?
ESH and EH
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An increase in temperature will...
...increase the rate of reaction as more molecules will exceed activation energy, and number of collisions of enzyme with substrate will increase.
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if optimum temperature is exceeded...
...the enzyme will denature as the H bonds will be broken and protein will unfold.
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What type of inhibitors lie under the non-covalent regulation category?
1) Simple inhibitors- competitive (to enzyme), non-competitive (to enzyme and substrate) and uncompetitive (to substrate)2) Allosteric inhibition
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What type of inhibitors lie under the covalent regulation category?
Reversible modification and irreversible modification
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Describe competitive inhibition
has a structure similar to the substrate, and hence occupies the active site instead of the substrate, taking the free enzyme out of the reaction in a reversible manner. (I and S compete and their binding is mutually exclusive)
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Why does Vmax remain unchanged with the addition of a competitive inhibitor?
Because as [S] increases to infinity, all inhibitors will be displaced
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Why does the addition of a competitive inhibitor result in the curve shifting right and hence an increase in km
At all [S], inhibitor shifts equilibrium from ES to E. Hence ([E][S])/[ES] will increase, so Km will also appear to increase. This decreases the apparent affinity of the enzyme for the substrate
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Describe non-competitive inhibition
A molecule that doesn't have a similar shape to the substrate, but binds elsewhere than the active site. This changes the shape of the enzyme and the active site, meaning that the substrate can no longer fit. Therefore, no reaction occurs.
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Why is the effect not reversed by adding more S? Therefore?
Because it does not bind to the same place as the S. Km is unaffected.
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What will it reversibly bind?
The enzyme as well as the enzyme substrate complex.
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What happens to Vmax? Why?
It decreases as catalytic rate is also reduced
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Describe uncompetitive inhibition...
when the inhibitor binds only with the ES to make ESI. This complex cannot yield products
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What happens with increasing [S]?
It does not reverse the reaction
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Where is this type of inhibition usually found?
In enzymatic reactions with multiple substrates
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What appears to happen to Km?
It decreases i.e. there is an apparent increase in affinity of substrate to enzyme (but actually substrate release is just being inhibited)
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What happens to Vmax with increasing [I]? What about Km/Vmax?
It also decreases. It remains constant
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Describe allosteric inhibition
the effector binds at another site on the enzyme known as ‘allosteric site’ i.e. is non-competitive
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What is the difference between this and simple inhibition?
allosteric effectors can be positive or negative and are much more sensitive than simple inhibition. I.e. relatively small amounts of effector are required to have relatively large effects.
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What are they useful?
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What does the binding of the effectors do?
Binding of effector to allosteric site alters protein conformation. This change is transmitted to the active site. (activator improves substrate binding whilst inhibitor reduces it)
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Negative feedback is common with allosteric inhibitors. What is a feedback loop?
Refers to when downstream products regulate upstream reactions
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Allosteric proteins are oligomeric. What does this mean?
substrate binds one subunit and effector to the other
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What shaped curve do allosteric enzymes have?
Sigmoid (low [S] = low activity, middle [S] = a sudden rise, high [S]= levels off to Vmax)
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What way would an activator/inhibitor shift the sigmoid curve?
Activator - profile shifts left... more sensitive to amount of substrate. Inhibitor- profile shifts right... less sensitive to amount of substrate
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What does the sigmoid curve demonstrate?
1. cooperativity. i.e. substrate binding at one site can affect substrate binding at the next. 2. Small change in [I] can cause a large change in V -i.e. small curve shift but large effect on velocity.
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Is Vmax affected by inhibitors/activators? Why is the sigmoid curve different to that of M-M kinetics?
No. Because allosteric proteins do not follow the Michaelis-Menten kinetics.
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In what circumstances can allosteric regulation be considered insufficient?
When enzyme activity must be limited to certain locations it is produced in an inhibited state. -When activity must be switched off reversibly
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What is a sufficicent alternative?
Covalent modifications- can alter activity dramatically via activation or inactivation.
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Describe reversible covalent modification...
This is very sensitive- can change from 0% to 100% activation. It is a way of switching off an enzyme temporarily before switching it back on again.
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GIve an example
Phosphorylation of pyruvate dehydrogenase by ATP --> inactive enzyme
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When is irreversible regulation neccessary?
When enzymes are potentially hugely harmful e.g. proteases could digest all proteins!
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Describe irreversible regulation.
enzymes are synthesised as inactive precursors known as zymogens. These are recognisable by their name having ‘ogens’ on the end. They are then cleaved (end clipped off) which activates them.
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Give an example
Trypsin This is a digestive enzyme which attack proteins- if immediately active would digest the host cell (leading to pancreatitis). Therefore: Trypsinogen --- enterokinase---->trypsin + hexapeptide
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Card 2


are all enzyme controlled reactions reversible?


Yes- those that are considered irreversible are only because the product will diffuse off (but if it were trapped then it would be reversible)

Card 3


Why can we consider only the forwards reaction when looking at enzyme kinetics?


Preview of the front of card 3

Card 4


What is activation energy?


Preview of the front of card 4

Card 5


Give two ways in which the energy barrier can be overcome


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