enzymes Flashcards
what are enzymes
Crucial for life.
Accelerate chemical reactions under physiological conditions (body temperature, normally aqueous solution at neutral pH) without being used up.
Catalyst is never used up
Catalysts are reused
Catalyst helps reaction proceed faster
almost all enzymes structures are what type of protein
globular proteins
what are the 2 classes of enzyme globular protein structure
Simple - only protein. Amino acid side chains carrying out catalysis.
Conjugated – also need small non-protein components to function.
(Conjugated means something attached to something else)
enzyme protein takes shape due to what
amino acid side chains
what are the 2 types of conjugate enzymes
Apoenzyme – the protein component
Cofactors - non protein component
(Cofactor are non organic, so no carbons in structure)
Organic means they have carbon
describe cofactor enzymes
May be one or more inorganic ions such as Zn2+, Mg2+, Mn2+, Fe2+, Ni2+ or a complex organic molecule such as NAD called a coenzyme.
eg. Ni2+ is the cofactor in the enzyme urease.
eg. NAD is the coenzyme in the enzyme lactate dehydrogenase
Most vitamins act as coenzymes in the human body.
When a cofactor/coenzyme is bound to the enzyme it is also referred to as a “prosthetic group”.
what are the rules for naming enzymes
‘Common’ given suffix “ase” or “in” e.g. DNA polymerase; Pepsin
Systematic names e.g. Hexokinase has the systematic name: ATP: glucosephosphotransferase
Enzyme Commission numbering system based on
the reactions they catalyse e.g. EC3.4.11.4 are the
“Tripeptide aminopeptidases”
name the main groups of enzymes
Oxidoreductases (EC1)
Transferase (EC2)
Hydrolases (EC3)
Lyases (EC4)
Isomerases (EC5)
Ligases (EC6)
describe the enzyme group Oxidoreductases (EC1)
Act on many chemical groupings to add or remove hydrogen ions eg. oxidases, reductases, dehydrogenases
eg. dehydrogenase catalyses the detoxification of alcohol in the body.
describe the enzyme group Transferase (EC2)
Transfer functional groups between donor and acceptor molecules.
eg. transaminase transfer amino groups, kinases transfer phosphate groups
describe the enzyme group Hydrolases (EC3)
Hydrolysis of substrates with addition of water across the bond.
eg. lipases hydrolyse fats, proteinases and peptidases hydrolyse proteins
(This happens when we digest our food
when we ingest proteins)
describe the enzyme group Lyases (EC4)
Add water, ammonia or CO2 across double bonds, or remove these groups to produce double bonds.
eg. decarboxylases remove carboxyl groups as CO2
describe the enzyme group Isomerases (EC5)
Carry out many different types of isomerization reactions
eg. racemases interchange between D and L amino acids
(2 mirror images – isomers )
describe the enzyme group Ligases (EC6)
Catalyse reactions in which two chemical groups are joined (ligated) using energy from ATP eg. synthetases, carboxylases.
enzymes provide an environment which is what
is energetically favourable for a reaction to take place.
for a reaction to occur what might have to happen to substrate molecules
Substrate molecules may have to be brought close together and be orientated correctly in space in relation to each other.
why is energy needed for a reaction to occur
Energy is needed to overcome forces of repulsion between reacting molecules and to weaken existing chemical bonds.
what is the activation energy
i.e. work is required – activation energy must be put into the system, even for ‘spontaneous’ reactions to occur.
what the affect of an enzyme on activation energy
But less activation energy is required when an enzyme is involved.
how does an enzyme reaction achieve a lower activation energy
A catalyst, eg. an enzyme, provides an alternative route for the reaction with a lower activation energy
how doe enzymes impact equilibrium
As with chemical catalysts, enzymes cannot alter the equilibrium of reversible reaction.
Enzymes however can still speed up the reversible reaction
what does the active site provide
specific, high-affinity binding of substrate(s) and an environment that favours catalysis
in most enzymes how many active sites participate in the interaction with substrates.
1
explain how the crevice-like active site id formed
formed by R-groups from different parts of the protein chain(s) - brought together by folding and bending of the protein (3o level structure).
(Shape the active site takes depends on the R group attached )
(There are also other sites on an enzyme )
whats the active site usually structured to exclude, and whats it surrounded by
The active site is usually structured to exclude water – and is surrounded by non-polar R groups (unless water is involved in the reaction).
Its hydrophobic
whats the rest of the enzymes job, excluding the active site
to maintain the correct shape of the active site.
Anything changing shape of enzyme will affect its function occurring
what are the different type of enzyme substrate interactions
lock and key
induced fit
explain the lock and key model
In this model the active site has a fixed, rigid geometrical conformation.
Only substances whose shape and chemical nature are complementary to the active site can interact.
The shape of substrate and active site on enzyme have to compliment each other
explain the induced fit model
Many enzymes have flexibility in their shape.
Substrate binding induces a conformational change at the active site to give the correct fit
So is why many enzymes have flexibility in shape to fit the substrate
Evidence for this: The active site of glucose hexokinase changes
shape as it interacts with the glucose
substrate.
when does enzyme substrate complex form
when enzyme and substrate bind
explain hoe substrate molecules bind to enzymes and how the reaction occurs
The R groups at the active site are involved in binding the substrate by relatively weak:
Electrostatic interactions.
Hydrogen bonds.
Van der Waals bonds.
Hydrophobic interactions.
help reaction overcome the activation energy barrier
Catalytically active groups in the active site then act on the substrate to transform it into the transition state and then the product:
Orientation & Proximity Effects; Acid-Base Catalysis; Covalent Catalysis; Stabilisation of intermediates etc.
Active site groups will act on substrate to form enzyme substrate complex to form product
The enzyme-product (EP) complex then breaks down, releasing enzyme and reaction products
Enzyme doesn’t get consumed in reaction so is released as same to be reused
active site takes shape cos of what
hydrophobic groups
binding strength is ____ between substrate and enzyme
weak
describe enzymes and specificity
Enzymes differ in the degree of specificity that they show.
what is absolute specificity
Enzyme will only catalyse a particular reaction for only one substrate.
Uncommon
eg. urease - splits urea into ammonia and CO2
what is stereochemical specificity
Enzyme can distinguish between stereoisomers.
eg. arginase hydrolyses the L form of arginine but not the D form.
what is group specificity
Involves structurally similar compounds with the same functional groups.
eg. Carboxypeptidase cleaves amino acids, one at a time, from the carboxyl end of the peptide chain.
name the factors affecting enzyme catalysed reactions
temp
pH
substrate
enzyme
how does temp affect enzyme reactions
Plots of initial rate vs. temperature usually gives a bell-shaped curve.
In the same way as for inorganic catalysts, reaction rate increases as temperature is increased.
Enzymes + substrates have more kinetic energy – collide more often & molecules have energy to overcome the (reduced) activation energy.
Increase temp increases enzyme activity
But there’s an optimum temp for their activity.
After this optimum enzyme denatures – changing shape, and activity reduces
40 degrees is optimum temp
how can temp cause inactivation of enzymes
The onset of inactivation (denaturation) occurs over a few degrees, typically in the range 50-60oC but varies between enzymes.
whats the optimum temp for diff enzymes
Mammals: around 40oC
Thermophilic bacteria: 90oC
Artic snow flea: -10oC
what shape of curve does optimum ph give
Plots of enzyme activity vs. pH usually also give a bell-shaped curve.
what happens to charges if PH is messed about with
If mess about with PH you mess about with charges
This change causes enzyme shape to change
how does PH affect enzyme activity
pH affects the ionisation state (charge) of R-groups, possibly affecting the shape of the active site (3o level structure) as well as their participation in the enzyme reaction (at active site).
pH may also affect the ionisation state (charge) of the substrate.
Also, denaturation of the enzyme may occur at pH extremes.
give some examples of pH optima
pepsin (stomach) - 1.5-2.5
amylase (saliva) - 6.8
trypsin - 8.5
arginase (liver) - around 10
explain the relationship between enzyme and concentration
A linear relationship between the amount of enzyme and the rate of reaction (v) is often found.
As increase conc you increase the rate
As the conc. of substrate increases the velocity of the reaction _____ to a _______ when _______
increases to a maximum, Vmax when the enzyme is saturated with substrate, [ES] is at a maximum
Vmax = maximum velocity
what are international units, in terms of measurements of enzyme activity
International unit, per g or per ml, is the
amount of enzyme which transforms 1 mmole of
substrate in 1 minute.
what does specific activity indicate
Indicates the amount of enzyme in relation to the amount of protein in a crude enzyme extract (not pure).
expressed as: mmol/min/mg of protein or
units/mg protein
Useful for estimating the purity of an enzyme.
how can we measure activity
We must know the overall equation for the reaction,
We need a suitable ‘signal’ i.e. a means of measuring the progress of the reaction - often change in absorbance.
We must control the reaction conditions: temperature, pH, cofactors, substrate conc.
Substarte should reduce and products formed should increase
what is Vmax
the maximum speed of an enzymatic reaction.
Enzyme is working at full capacity & active sites are saturated.
Each enzyme has a characteristic Vmax
what do we do to determine the maximum speed of an enzymatic reaction,
the substrate concentration is increased until a constant rate of product formation is achieved (Vmax).
what can Michaelis-Menten constant, KM, be defined as
the concentration of substrate at half the maximum velocity.
Has units of concentration and is independent of enzyme concentration.
Enzymes are further characterized by the substrate concentration at which the rate of reaction is half Vmax – this value is KM
how do you work out the KM
half of Vmax, go along and then down graph
in terms of Michaelis Menten curve what does the speed v mean
the number of reactions per second that are catalyzed by an enzyme
what happens with increasing substrate concentration (s) in Michaelis Menten curve
With increasing substrate concentration [S], the enzyme is asymptotically approaching its maximum speed Vmax, but never actually reaching it. Because of that, no [S] for Vmax can be given. Instead, the characteristic value for the enzyme is defined by the substrate concentration at its half-maximum speed (Vmax/2). This KM value is also called Michaelis-Menten constant.
explain albinism
Genetic.
Often caused by the absence of the enzyme tyrosinase.
Tyrosinase is necessary for production of the pigment melanin.
In classic albinism tyrosinase is absent or in reduced amounts.
Role of enzyme is to produce pigment melanin
what does Km give an indication of
the affinity of an enzyme for its substrate.
what does a high Km mean
low affinity
what does a low Km mean
high affinity
so binds more strongly.
what is catalase and its Km value
is a common enzyme found in living organisms. Its functions include catalyzing the decomposition of hydrogen peroxide to water and oxygen. Catalase has one of the highest turnover rates for all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. 2 H2O2 → 2 H2O + O2
KM = 0.025 M
what is glucose isomerase and its Km value
is an enzyme that catalyzes the conversion of glucose into fructose. In the glycolysis pathway phosphoglucose isomerase converts glucose-6-phosphate to fructose-6-phosphate. These reactions are reversible.
KM = 0.4 M
explain the difference between catalase and glucose isomerase Km value
Catalase has the smaller KM therefore less substrate is needed to effectively catalyse the formation of the product.
Catalase has a greater affinity for its substrate.
explain Lineweaver-Burk Plot
offers an improved graphical means of determining KM and Vmax
KM and Vmax are determined from the intercepts on the x and y-axes.
explain what enzyme deficiencies are
Deficiency in enzyme function are often a result of a genetic disorder.
Normally enzyme deficiencies lead to the accumulation of specific intermediary metabolites in plasma and hence in urine
name enzyme deficiencies
lactose intolerance
Phenylketonuria (PKU)
Albinism
explain how lactose intolerance occurs from enzyme deficiency
Shortage of the enzyme lactase, which is produced by the cells that line the small intestine.
explain how Phenylketonuria (PKU) occurs from enzyme deficiency
The amino acid Phenylalanine is normally converted to the amino acid tyrosine by the enzyme Phenylalanine hydroxylase as part of normal amino acid metabolism.
People with PKU lack this enzyme resulting in a built-up of phenylalanine in the body.
explain how albinism occurs from enzyme deficiency
Genetic.
Often caused by the absence of the enzyme tyrosinase.
Tyrosinase is necessary for production of the pigment melanin.
In classic albinism tyrosinase is absent or in reduced amounts.
what are tests for enzyme deficiency
Eye exam
Testing of hair root for tyrosinase & any resulting pigment.
what are some effects of enzyme deficiency
Varied condition.
People may have little or no pigment in their eyes, skin, or hair.
Usually people have problems with vision.
what do enzyme inhibitors do
A large variety of compounds slow down or stop the normal catalytic function of enzymes by binding specifically to them.
where are enzyme inhibitors found
Most poisons.
Some produced for defence.
Many drugs.
May include metabolic waste products.
Some produced for controlling metabolism.
Present in some foods
explain enzyme inhibitors
Some help control metabolism –naturally present in cells and are used to control metabolism.
Inhibitors may act to poison the metabolism of susceptible creatures eg. Antibiotics
Metabolic waste can serve as enzyme inhibitors, especially those found in environments which are rarely refreshed ie. Fermentation.
what are the main types of inhibition
- Reversible
competitive
non competitive
Uncompetitive - Irreversible
what are the 3 types of reversible inhibition
competitive
non competitive
uncompetitive
describe some Shared features of reversible inhibition
When concentration of inhibitor drops, enzyme activity is regenerated.
Usually bind to enzymes by weak, non-covalent forces.
The inhibitor maintains a reversible equilibrium with the enzyme.
explain competitive inhibition (reversible)
Both inhibitor and substrate bind at the active site on a random basis.
The inhibitor effectively competes with the substrate for the active site.
Typically have a similar shape/structure to normal substrate molecules but are unable to take part in the reaction.
Competitive inhibitor – temporarily blocks the active site
what affect does the addition of substrate have on competitive inhibition
By raising the substrate concentration sufficiently the effect can be overcome and the maximum velocity can be reached.
explain glucose and invertase in terms of competitive inhibition
Often include the products of the reaction – a form of biological control.
Glucose is a competitive inhibitor of the enzyme invertase.
explain non-competitive inhibitors (reversible)
Bind non-covalently and reversibly to a site other than the active site.
Changes the 2prime and 3prime structure of the enzyme, including that of the active site, reducing its ability to function.
Binds to E or ES
The degree of inhibition depends on the concentration of the inhibitor present.
The enzyme may bind with inhibitor, substrate or both at the same time.
E = enzyme
Es= enzyme substrate
whats the affect of substrate addition on non-competitive inhibitors
Adding more substrate cannot completely reverse the effect and the maximum velocity is decreased proportionately to inhibitor concentration.
explain uncompetitive inhibition (reversible)
A rare form where the inhibitor binds only to the ES complex, slowing/preventing catalysis. A number of drugs and pesticides are thought to operate in this way.
The binding site for the inhibitor is accessible only after the enzyme has bound to its substrate.
Lithium, used as a mood-stabiliser in e.g. bipolar disorder, is thought to work as an uncompetitive inhibitor.
explain irreversible inhibition (inactivators)
Form strong covalent bonds with amino acid side chains of the active site of the enzyme, permanently deactivating it.
Inhibitors do not have to be structurally similar to substrate molecules.
Permanently prevents substrate from occupying site, therefore there is no reaction.
how to identify inhibition type
competitive = Km is increased: inhibitor interferes with binding of the substrate to the enzyme. Vmax is unaffected: inhibitor doesn’t interfere with catalysis in the enzyme-substrate complex
uncompetitive= Km is decreased: inhibitor binds to and stabilises the ES complex, making it more difficult for S to dissociate, giving an apparent increase in enzyme affinity for S.
Vmax is decreased: inhibitor interferes with catalysis in the enzyme-substrate complex
non-competitive = Km is unaffected: inhibitor doesn’t compete for substrate binding site. Vmax is decreased: inhibitor interferes with catalysis in the enzyme-substrate complex
name ways to control enzyme activity
Compartmentalisation
Allosteric enzymes
Zymogens
Covalent modification
explain Compartmentalisation of Enzymes
Enzymes involved in a particular metabolic pathway may be compartmentalised in specialised organelles.
Optimal conditions for individual enzyme catalysed reactions are more easily achieved
explain lysosomes in terms of compartmentalisation of Enzymes
Lysosomes are packed with a mixture of hydrolytic enzymes.
They engulf and breaking down bacteria, which have entered the cell as well as cell debris etc.
The lysosomes have sometimes been likened to “The Police Force of the Cell”. Even in a place as small as a cell, we need someone to keep things in order. But unlike the police, these lysosomes literally eat things which disturb the natural order of the cell. Lysosomes pick up foreign invaders such as bacteria, food and old organelles and break them into small pieces that can hopefully be used again. If they pick up a really harmful invader, they will eat it up and expel what is left of it out of the cell so that the debris can be removed from the body.
The lysosome is able to do this because it is filled with enzymes. These enzymes are specially made for the lysosome by the rough endoplasmic reticulum and work only at low pH (highly acidic) levels. The reason for this is that the enzymes are so strong that they could eat the whole cell if the lysosome ever let them out. However because they can only work at low pH levels and the rest of the cell has a neutral pH level, they can be neutralized if they accidentally escape from the lysosome.
what are lysosome enzymes
The enzymes could ‘digest’ the cell if they accidentally escaped from the lysosome.
However they work only at low pH (optimum pH 4.5 – 5.0)
The rest of the cell has a neutral pH (pH 7.0). This protects cell contents from any of the lysozyme enzyme that may be released.
explain enzyme and metabolism
In every metabolic pathway, the activity of at least one enzyme is subject to regulation so that the flux of material through the pathway can be controlled.
ability of enzymes to be controlled is of no less importance than their catalytic activity
name REGULATION OF ENZYME ACTIVITY BY CELL COMPONENTS examples
Allosteric Control
Zymogens
Covalent Modification
explain allosteric control
The activity of some enzymes is inhibited or promoted by the binding of small, physiologically important molecules that are not their substrates or products.
explain structure of allosteric enzymes
Allosteric enzymes are always composed of subunits i.e. have 4o structure (2 or more protein chains).
They have 2 types of binding site: those for substrate and those for regulators - distinct in location (often on different proteins) and shape.
what are regulators in terms of allosteric enzymes
and explain the pos and neg effectors
Regulators cause conformational changes that are transmitted through the bulk of the protein to the active site.
POSITIVE effectors – increase catalytic activity
NEGATIVE effectors – reduce/inhibit catalytic activity
describe kinetics in terms of allosteric enzymes
Allosteric enzymes often show sigmoid graphs of velocity (initial rate) vs. [S]
Sigmoid because substrate binding is “cooperative.” i.e. binding of first substrate at the first active site stimulates active shape, and promotes binding of second substrate
what is feedback control
Feedback control is one of the most common mechanisms, by which allosteric enzyme activity is regulated.
Activation or inhibition of the first reaction in a sequence of reactions within a cell is controlled by what
a product of the reaction sequence.
explain the feedback control reaction sequence
Each step is catalysed by a different enzyme.
The product of each step is the substrate for the next enzyme.
Final product (D) is a negative regulator enzyme 1:
At low concentrations of D, the reaction sequence proceeds rapidly.
At higher concentrations of D, the activity of enzyme 1 becomes inhibited by feedback, and eventually the activity stops.
Later when the concentration of D decreases through its use in other reactions, the activity of enzyme 1 increases and more D is produced.
name an Example of Allostric Control and Feedback Inhibition
Aspartate transcarboxylase (Atcase)
what is Aspartate transcarboxylase (Atcase)
CTP catalyses the production of Carbamyl aspartate, in a series of reactions in the production of the pyrimidine nucleotide triphosphates (for biosynthesis of DNA/RNA).
Atcase structure
consists of six catalytic subunits and six regulatory subunits.
what does Binding of CTP to sites in regulatory subunits change and what graph does this result in
changes conformation, decreasing binding of substrate to active site
This results in a sigmoid curve when initial velocity is plotted against aspartate concentration. due to The binding of the two substrates aspartate and carbamyl phosphate is cooperative hence sigmoid curve.
how does the Allosteric Control and Feedback Inhibition (ATcase) reaction have sensitivity
The curve has a steep section in the middle of the substrate concentration range, reflecting the rapid increase in enzyme velocity, which occurs over a narrow range of substrate concentrations. This allows allosteric enzymes to be particularly sensitive to small changes in substrate concentration within the physiological range.
what does ATP have to do with Allostric Control and Feedback Inhibition - ATcase
ATP, produced earlier in the pathway acts as an allosteric activator, increasing affinity of the enzyme for its substrate.
ATP acts at the same binding site as CTP (ie. competes).
High levels of ATP signal to the cell that energy is available for DNA replication, and so ATCase is activated, resulting in the synthesis of the required pyrimidine nucteotides. When pyrimidines are abundant, the high levels of CTP inhibit ATCase, preventing needless synthesis of N-carbamyl aspartate and subsequent intermediates in the pathway.
what do zymogens do
Some enzymes have the potential to destroy the tissues that produce them. They are therefore generated in an inactive form and then converted to an active form when needed.
give examples of zymogens
most digestive enzymes and blood-clotting enzymes.
what are The inactive forms (inactive precursors) of an enzyme are called
zymogens or proenzymes.
what happens to zymogens once secreted
theyre irreversibly converted to their active form.
how are zymogens activated
Activation requires an enzyme-controlled reaction that either adds to the zymogen structure or removes a part of it.
Most commonly involves removal of a segment of polypeptide chain.
how is trypsinogen secreted
Trypsin is secreted as an inactive precursor trypsinogen in the pancreatic juice. Once in the duodenum it is acted-on by enteropeptidase, secreted by the cells in the duodenal wall.
Trypsin goes on to activate further trypsinogen molecules and other protease precursors.
explain covalent modification
Enzyme control through the making and breaking of a covalent bond between a non protein group and an enzyme molecule.
The most common example is the addition/removal of a phosphate group.
About 20% of enzymes employ this.
Reversible process
The negative charge attracts positively charged amino acid side-chain groups so its addition to or removal from a protein will change the characteristics of the protein.
Often this is a conformational change.
what is molecular switch in terms of covalent modification
If the two conformations give the protein different activities (i.e. being enzymatically active in one but not in the other) addition/removal of phosphate acts as a molecular switch.
Rapid and easily reversible.
what is phosphorylation
catalysed by a large group of enzymes termed kinases (+PO4)
what does protein kinase do
take a phosphate off ATP and place it onto an amino acid side chain of a protein:
explain dephosphorylation
catalysed by a large group of enzymes termed phosphatases (-PO4)
