enzymes Flashcards
(110 cards)
1
Q
what are enzymes?
A
- biological catalysts that…catalyse nearly all of life’s reactions. Therefore, they are one of the major drug targets
2
Q
if we want to change the rate of a chemical reaction in the body to treat a disease then it is desirable to change what?
A
the catalytic activity of an enzyme.
3
Q
- how do we know a lot about enzyme structure and mechanics:
A
these can be isolated and studied in vitro
4
Q
why are enzymes important
A
- inhibitors of: proton pumps, phosphodiestertases, reductases, kinases
- herbicides
- pesticides
- DNA methylation / histone modification
5
Q
key points of enzymes as catalysts
A
- Specificity
- Mechanism of action
- Chemical identity
6
Q
rules of catalysts (5)
A
- A catalyst cannot catalyse a thermodynamically unfavourable reaction.
- A catalyst cannot change the course of a reaction.
- A catalyst cannot change the equilibrium of a reaction, only the rate at which
equilibrium is reached. It lowers the activation energy for the reaction. - A catalyst may exert a directing influence. If two reactions are thermodynamically
possible (A going to B or C) and a catalyst only catalyses one of them, then that
reaction will be favoured. - A catalyst is recoverable, so only small amounts are necessary.
7
Q
Explain how enzymes can act as catalysts for biological reactions.
A
- For a reaction to occur there must be a release of energy (exothermic).
- Enzymes catalyse reactions by lowering the activation energy required for a reaction to pass through its transition state.
This can involve
- Positioning substrates precisely for reaction (orbital steering)
- Formation of an unstable covalent intermediate (transition state) that more readily reacts
- Increasing effective concentration of reactants by binding at active site
- Binding substrates to bring them into proximity with catalytic groups
- Inducing strain or distortion in a susceptible bond
8
Q
Ordinary chemical catalysts will usually speed up a type of reaction irrespective of the nature of the compound involved. How are enzymes different?
A
enzymes are often specific for one reaction only and will even distinguish between optical isomers (although the degree of specificity of enzymes varies).
Since many different reactions occur in living systems and the enzymes are specific, a lot of different enzymes are necessary for the systems to function.
9
Q
some reactions are universal → about (…) enzymes are commonly found in all organisms.
A
200
10
Q
catalysis occurs where?
A
at the active site of the enzyme and the tertiary protein structure that determines the 3D shape of the enzyme is absolutely essential for its specific binding of substrates and its catalytic function.
11
Q
it was previously thought that enzymes are always proteins, but it is now known that some (…) also possess catalytic activity.
A
nucleic acids
12
Q
However, in general, the properties of enzymes are those associated with the structure of proteins i.e., their function depends on their shape which is determined by tertiary structure and held together by non-covalent bonds. What leads to loss of enzyme catalytic activity
A
Disruption of these bonds (e.g. by extremes of heat or pH) which is termed DENATURATION
13
Q
mechanism of action:
- why are enzymes so good?
A
- Enzymes increase the rate of the reaction they catalyse more than simple chemical catalysts do.
- For example, Urease increases the rate of hydrolysis of urea by a factor of 10^14
14
Q
The catalytic power of an enzyme is the ratio of
A
enzyme-catalysed rate of reaction to uncatalysed rate.
15
Q
For a reaction to occur there must be a release of energy (exothermic). Before a substrate can achieve this in its formation of products it must first pass through a higher energy state called
A
the transition state. (To do this it must acquire activation energy. An enzyme acts as a biological catalyst by lowering this activation energy)
16
Q
the enzyme lowers the activation energy for the reaction by doing what?
A
binding the substrate(s) in its active site.
17
Q
- what effect can binding the substrates in its active site have?
A
- The enzyme may position substrates in its active site and line up molecules precisely for reaction so that. bonds can be broken or made more easily (“orbital steering”).
- Some enzymes combine with the substrate to form an unstable covalent intermediate in a “transition state” that more readily undergoes reaction to form products. Substrates in the active site are oriented such that a transition state is readily formed.
- Binding substrates in the active site increases the effective concentration of the reactants.
- Binding substrates in the active site may bring them into close proximity to a catalytic group. Enzymes may provide functional groups capable of acting as proton donors or proton acceptors, and an enzyme may bring about general acid - base catalysis.
- Metal ion catalysis is also a method used by some enzymes.
- General acid - base catalysis is the most ubiquitous sort of reaction, being found in the reactions of the dehydrogenases, the serine proteases, thiol proteases and carboxypeptidase, ribonuclease and lysozyme. - The enzyme may induce strain or distortion in the susceptible bond. There is much evidence for the distortion of the enzyme on binding of substrate. These changes of shape are called conformational changes.
18
Q
what forms the active site
A
- The enzyme protein has 3D shape from its tertiary structurethat determines its active site (the site of catalysis).
- The amino acids forming the active site are few and may be far apart in the primary sequence but are spatially close due to the 3D structure.
19
Q
The functional groups in the side chains of these amino acids interact with the substrate usually through weak non-covalent forces such as ionic bonds, hydrogen bonds and hydrophobic bonds and position what?
A
the substrate within the active site
20
Q
A transition state is formed between the enzyme and substrate (ES) which will then do what?
A
regenerate free enzymes plus products.
21
Q
how to work out the name of an enzyme
A
= substrate + reaction + ‘ASE’
enzyme
substrates —————> products
22
Q
The recommended systematic enzyme nomenclature is based on the type of reaction the enzyme catalyses, e.g.
- for oxidoreductases (or dehydrogenases) e.g. lactate dehydrogenase
A
– catalyse oxidation and reduction reactions.
23
Q
Transferases e.g. aspartate aminotransferase
A
– transfer functional groups from donor to acceptor
24
Q
Hydrolases e.g. esterases (acetylcholinesterase), proteases
A
hydrolysis of C-O, C-N and C-C bonds
25
Lyases e.g. pyruvate decarboxylase
break bonds such as C-C, C-O, C-N.
26
isomerases e.g. alanine racemase
geometric or structural changes within a molecule.
27
Ligases e.g. glutamine synthetase
– joins molecules together, forms bonds, requiring ATP energy.
28
enzyme assays: (measurement of activity)
- after extraction/purification of an enzyme from a tissue, its reaction can be studied in vitro: how?
by mixing enzyme and substrate together under suitable conditions, and following
- the disappearance of substrate
- the appearance of product
29
what is an appropriate analytical technique to check this measurement of enzyme activity
colourimetry
30
what factors affect enzyme activity:
1. time
2. enzyme concentration
3. temperature
4. pH
5. cofactors (presence or absence of them)
6. substrate concentration
31
Describe FIVE (5) different factors, other than the presence of an inhibitor, that affect enzyme activity.
1.**Time**: Most reactions are linear initially (initial velocity) before slowing down due to decreased substrate concentration or increased product concentration (which may inhibit the reaction / increase rate of reverse reaction / change pH etc).
2.**Temperature**: Rate increases with temperature like all chemical reactions. However high temperature denatures most proteins. Therefore the amount of product produced will tend to decrease above a certain temperature.
3.**pH**: Enzymes active over a limited range of pH, and, in most cases, a definite pH optimum is observed. Many ionisable groups present, so ionisation may affect shape and binding at active site. Extremes of pH may denature the enzyme
4.**CoFactors**: A component that is required in addition to enzyme and substrate. Coenzymes are complex organic molecules derived from water-soluble vitamins. Other cofactors could be inorganic ions or activators
5.**Substrate (and Enzyme) concentration**: Enzyme activity increases with substrate concentration showing saturation kinetics / rectangular hyperbola. This relationship can be described in terms of the Michaelis-Menton equation (v = Vmax[s] / Km + [s]). Generally reaction rate increases linearly with enzyme concentration.
32
how does time affect it?
1. Time
The velocity of an enzyme reaction is the change in reactant or product concentration with time. Usually
appearance of product is measured, so the velocity = appearance of product with time.
The initial velocity is the tangent to velocity at time 0. Most reactions are linear for some time before tailing off.
The slowing down of the reaction is due to:
* a) The concentration of substrate decreases.
* b) The concentration of product increases, and this may:
* inhibit the reaction
* increase the velocity of the reverse reaction
* cause a change in pH and slow the reaction
33
Factors affecting enzyme activity
2. Enzyme concentration
* Within limits, the more enzyme present the faster the reaction goes.
* If the product inhibits the enzyme then there may be a limit.
* The amount of enzyme present in a tissue sample can be calculated from an assay of
activity assuming that this linear relationship holds. This is important in clinical
measurements (e.g. creatine phosphokinase in blood).
34
Factors affecting enzyme activity
3. Temperature
* The rate of an enzyme catalysed reaction increases with temperature like all chemical reactions.
* However, high temperatures denature most proteins.
* Most enzymes are inactivated usually between 60-70oC (exceptions are enzymes of bacteria
which live at high temperatures in hot springs).
* Therefore, although initial velocity increases with temperature, the actual amount of product
decreases above a certain temperature.
35
Factors affecting enzyme activity
4. pH
* Enzymes are only active over a limited range of pH and in most cases a definite pH optimum is
observed.
* Since enzymes are proteins containing many ionisable groups, they exist in a whole series of
different states of ionisation which depend on pH. This will have its greatest effect in the active site
where ionization may affect shape and any ionic binding of substrate. The ionization of the
substrate(s) must also be considered.
* Hence the relatively narrow range of pH within which the enzyme is active.
* Extremes of pH will denature the enzyme and so destroy activity.
36
Factors affecting enzyme activity
5. Presence or absence of cofactors
In many cases an additional component besides the enzyme and substrate is required in order that
the reaction may proceed. This cofactor could be:
* Organic coenzymes or prosthetic groups e.g. coenzyme A, NADH
* Inorganic ions or activators e.g. Mg2+, Mn2+, Zn2+, Cu2+ etc.
37
what are coenzymes
complex organic molecules
derived from water-soluble vitamins.
Cofactors take part in the chemical reaction
catalysed by the enzyme, often as carriers
of a particular chemical group e.g. COO- or
H+ or CH3.
38
Factors affecting enzyme activity
6. Substrate concentration
When initial velocity is plotted against substrate
concentration, the graph obtained is a rectangular hyperbola.
Thus initial velocity tends towards a maximum, Vmax (i.e.
when all available enzyme is saturated with substrate).
The value of Vmax will vary with the concentration of enzyme
used.
Enzymes show saturation kinetics. Notice that this plot does
not plateau out abruptly – it approaches Vmax hyperbolically.
It is NOT possible to calculate a true value for Vmax from this
plot because Vmax is achieved only at infinite substrate
concentration. Therefore a linear plot (Lineweaver-Burke) is
used.
39
what was the first reaction between an enzyme and a substrate
to form an enzyme - substrate complex and this was used by Michaelis and Menten (1913) to explain how enzymes show saturation kinetics.
40
what does enzyme kinetics quantify
the behaviour of an enzyme catalyst
41
Michaelis and Menton derived an equation that uses what assumption?
equilibrium assumption: so that the formation of a noncovalent enzyme-substrate complex is instantaneous, and that the breakdown to enzyme and products can be treated as a separate reaction
42
enzyme substrate equilibrium equation
E + S <-> ES <-> E + P
* k1 rate constant for formation of ES
* k -1 rate constant for conversion of ES to E + S
* k2 rate constant for product formation
43
what is the michaelis menten equation to do with initial velocity
v = V max [S] / km + [S]
44
what is Km (the michaelis constant)
* Km is a dissociation constant for a given enzyme
* Km is the concentration of substrate which allows the catalytic reaction to
proceed at 0.5 Vmax.
* 1/Km is a measure of the affinity between enzyme and substrate.
45
what is V max
Vmax is a constant for a given enzyme.
* Vmax is a quantitative assessment of the extent of catalysis
* Vmax is the theoretical maximal rate of the reaction. But it will never be
achieved.
46
measuring Km and Vmax using equations examples
https://www.notion.so/enzymes-1c800bb3982d80e984c4dbdedd3bfd12?pvs=4#1c900bb3982d80f7baf8ec04df0a9f56
47
why do we study enzyme kinetics
Km is the concentration of substrate which allows the catalytic reaction to proceed at 0.5 Vmax.
For a given enzyme Km is a constant and is independent of enzyme concentration.
Km is a dissociation constant, and 1/Km is a measure of the affinity between enzyme and substrate.
Vmax is a quantitative assessment of the extent of catalysis. Vmax is proportional to enzyme
concentration.
Where the actual enzyme concentration [E] is known:
The turnover number (kcat) gives the rate constant for the catalytic reaction. Enzyme turnover
numbers range from 10 - 107 per sec. An enzyme with a turnover number of 1000 sec-1 will catalyse
the conversion of 1000 molecules of substrate to product per second.
These parameters are therefore useful in the characterisation of a particular enzyme.
48
what assumptions do we make when calculating Michaelis-Menten enzyme kinetics
1. Enzyme - Substrate complex is formed.
This is now supported by overwhelming evidence, including X-ray crystallography.
2. That the equilibrium where the rate of ES formation is equal to its breakdown is reached instantaneously.
3. That the concentration of substrate in ES is negligible. Enzyme concentration will probably be about 0.001% of the substrate concentration, so the proportion of substrate locked up in the ES complex is very small.
4. There is no k-2 i.e. there is no chance of E + P forming ES – it is an irreversible reaction
49
allosteric enzymes have what distinct sites?
distinct regulatory (allosteric) and catalytic
50
binding of an effector (allosteric modulator) can lead to what effects on enzyme activity
stimulate or inhibit it
51
what about them having allosteric distinct sites from the catalytic active site allow?
allows the binding of an activator or inhibitor molecule (effector) which is structurally unrelated to the substrate and which can either stimulate or inhibit the enzyme activity by changing the shape of the active site and thus changing its affinity for substrate.
52
what could the effector be
another molecule in the metabolic pathway or even an ion such as Ca2+
53
in some cases, the regulatory and catalytic sites are where?
on different types of subunit e.g. aspartate transcarbamylase.
→This enzyme has six catalytic subunits and six regulatory subunits.
54
allosteric meaning
relating to or denoting the alteration of the activity of an enzyme by means of a conformational change induced by a different molecule.
55
what is co-operative binding?
when the binding at one site influences the binding of other sites e.g. conformational changes
56
pos vs neg co-operativity
- Positive Co-operativity: Binding of the first ligand increases the affinity of the enzyme for more ligands (e.g., hemoglobin with oxygen).
- Negative Co-operativity: Binding of the first ligand reduces the affinity for subsequent ligands.
57
what is an isoenzyme or isozyme
An enzyme which has multiple molecular forms (in the same organism) catalysing the same reaction
58
example lactate dehydrogenase (LDH): how many forms of this are there
five possible forms in organs of vertebrates.
→ is a tetramer made from two types of monomer - M (muscle), H (heart): these 2 in diff combos give the five isoenzymes
The two types of subunit have the same molecular weight but different amino acid compositions and are the products of two separate genes.
59
LDH forms
- LDH-1 (4H) - in the heart
- LDH-2 (3H1M) - in the reticuloendothelial system
- LDH-3 (2H2M) - in the lungs
- LDH-4 (1H3M) - in the kidneys, placenta and pancreas
- LDH-5 (4M) - in the liver and striated muscle
If LDH-1 level > LDH-2 level, myocardial infarction
(damage to heart tissues releases LDH-1 into the bloodstream).
60
what are multifunctional enzymes
have multiple active sites on a single polypeptide chain.
61
- example of a multifunctional enzyme
Mammalian fatty acid synthase
62
how many different catalytic activities does the enzyme above have?
has seven different catalytic activities in one protein molecule. This allows a metabolic pathway to operate within a single enzyme which has advantages in terms of speed and
control as well as absence from interference.
63
in multi enzyme complexes there is an association of separate enzymes in separate subunits e.g.
like different polypeptides
64
how are enzymes organised physically
so that the product of one becomes the substrate of another, without leaving the complex.
65
why are enzymes organised physically
This ensures a highly efficient progression from reactants to products. Such complexes protect the intermediates from competing reactions taking place in metabolism which would channel them away from making the desired product
66
enzyme inhibition:
- how do many drugs exert their action
by inhibition of enzyme activity in the bod
67
enzymes are biological catalysts it is possible to slow down certain cell processes by doing what?
the inhibition of a specific enzyme, and correct a disease state.
68
if the activity of the enzyme is vital to the cell/organism, then inhibition may lead to
the death of the cell or organism
69
note: Destruction of enzyme activity by protein denaturation (e.g., by strong acids or bases) is not regarded as
enzyme inhibition
70
reversible inhibitors: E + I ↔ EI
→ effect of the inhibitor is instantaneous
→ can be removed from the enzyme by dialysis or dilution so that the enzyme activity is returned to normal.
Such inhibitors react with the enzyme by weak non-covalent bonds to form an enzyme inhibitor complex
71
irreversible inhibitors: E + I → EI
→ The effect is therefore progressive, reaching a maximum when all the enzyme has reacted.
→ This is not easily reversed by simple physical treatments such as dialysis.
Bind very tightly to the enzyme, sometimes by formation of covalent bonds to form an enzyme inhibitor compound rather than a loose complex.
72
competitive reversible enzyme inhibition:
- why is the binding of the substrate and inhibitor mutually exclusive
Molecules which closely resemble the substrate in size, shape and charge distribution may also fit into the active site of the enzyme.
73
why might molecules which closely resemble the substrate result in inhibition?
because the active site is blocked. The inhibitor has a separate equilibrium with the enzyme.
74
Each of these equilibria is characterised by a dissociation constant. which ones?
the first by Km (the Michaelis constant) and the second by Ki which characterises the binding between enzyme and inhibitor.
75
Both I and S compete for the same active site: what effect does this have?
- if [S] is increased sufficiently while [I] is constant, the proportion of I bound to E will decrease, hence formation of ES will increase and the rate of enzyme action will increase.
- If sufficient [S] is present then eventually the inhibition by I will be overcome.
76
need to look properly at how can we represent reversible competitive enzyme inhibition on a graph + all the block toggles
https://www.notion.so/enzymes-1c800bb3982d80e984c4dbdedd3bfd12?pvs=4#1c900bb3982d8038a315d72d3ab24ec8
77
for an inhibitor to work as a drug in vivo, it must have what?
potency
specificity
78
- what do we mean by potency
the inhibitor will need to be potent enough so that the dose required is in the order of milligrams to grams
79
what do we mean by specificity
if a compound is a nonspecific enzyme inhibitor it is more likely to be toxic and exhibit side effects
80
how does a simple reversible inhibitor work?
binds to the enzyme and decreases activity instantaneously, and reverses within the time of enzyme action: the inhibitor binds non-covalently (ionic interactions/ h bonds) to the enzyme, and the strength of binding is of a similar order to the substrate
81
simple reversible inhibitor Ki vs Km size?
similar size
82
are simple reversible inhibitors potent enough
nah, not to work in vivo, where competition with the substrate occurs in a dynamic metabolic situation
83
when might they be?
when one which is a conformationally restricted competitive inhibitor, will have a higher affinity for the enzyme than the substrate, and hence is potent enough to work in vivo at reasonable concentrations: these might have Ki values in the region of 1x10^-7 mol/l
84
What are quasi-irreversible inhibitors?
A class of inhibitors that form long-lasting but not completely irreversible enzyme-inhibitor complexes.
85
What are tight binding inhibitors?
Competitive inhibitors that are conformationally restricted / form strong noncovalent interactions, leading to long-lasting complexes.
86
How strong is the binding of tight binding inhibitors?
They have very low inhibition constants (Kᵢ in the order of 10⁻⁹ to 10⁻¹⁰ mol L⁻¹), making them potent enough for in vivo drug use.
87
What are transition state analogues?
Inhibitors that mimic the transition state of a substrate and bind much tighter than standard substrate analogues.
88
Why are transition state analogues effective inhibitors?
They achieve very low Kᵢ values, sometimes in the nanomolar range, making them highly potent enzyme inhibitors.
89
How do some enzymes form covalent intermediates in their mechanism?
Certain enzymes, like acetylcholinesterase, can convert a compound into a slowly dissociating intermediate.
90
What characterises a slowly dissociating intermediate?
It results in time-dependent inhibition that is virtually irreversible.
91
how long can a slowly dissociating intermediate last?
it can persist for minutes or hours, compared to the normal enzyme mechanism where intermediates last only milliseconds.
92
What are some examples of quasi-irreversible inhibitors?
Anticholinesterases like neostigmine and physostigmine (eserine), as well as penicillin.
93
what are the 3 levels of regulation of enzyme activity within cells
1. Allosteric regulation
2. Covalent modification
3. Regulation of enzyme concentration
94
Some enzymes have binding sites for small molecules (effectors). The effectors can:
* Stimulate enzyme activity (activators)
* Inhibit enzyme activity (inhibitors)
These allosteric binding sites are distinct from the active catalytic site of the enzyme
95
what is feedback inhibition
Allosteric inhibitors are often end-products of a pathway that feed-back and inhibit an early step in the pathway to prevent overproduction and wastage.
96
what is feedforward activation
Allosteric activators are often substrates for the reaction or the pathway that feed-forward to encourage the pathway to dispose of the substrate.
97
mechanism of feedforward activation
1. The allosteric effector binds specifically and reversibly to the allosteric site(s) on the enzyme. The effector need not resemble the substrate in any way as the allosteric site is not the active site of the enzyme.
2. Allosteric binding is via non-covalent bonding (e.g. H-bonds, ionic interaction, hydrophobic interaction). Binding at the allosteric site induces a conformational change in the protein that alters the shape of the active site. Thus the important amino acid functional groups within the active site that are involved in binding and catalysis will shift position relative to one another.
3. This will either make it easier (activation), or more difficult (inhibition), to bind substrate and catalyse the reaction. When the effector dissociates from the enzyme the confirmation, and thus enzyme activity, returns to its original state.
98
some allosteric effectors can alter the Vmax of their target enzymes, but what do most alter to produce their effect?
they alter the affinity of the enzyme for its substrate
99
allosteric regulation and co-operativity:
- Many regulatory enzymes = multimeric meaning:
with several copies of a single polypeptide chain (subunit) each with its own active site, forming the quaternary structure of the enzyme.
100
in many cases, the binding of substrate to the active site of one subunit causes?
a conformational change that is transmitted through the quaternary structure to the other subunits and makes it easier for these to bind substrate: cooperativity
101
what do allosteric activators vs inhibitors do
allosteric activators stabilise the conformation of the substrate bound form
- allosteric inhibitors stabilise the conformation of the no substrate bound form
102
what does co-operativity allow an enzyme to do?
respond to small changes in substrate concentration with large changes in catalytic activity.
103
example of co-operativity
https://www.notion.so/enzymes-1c800bb3982d80e984c4dbdedd3bfd12?pvs=4#1c900bb3982d80ae8d5ff83e7efbdded
104
covalent modification
- an inactive precursor enzyme (zymogen or proenzyme) can be activated how?
extracellularly by proteolytic cleavage. E.g. chymotrypsinogen, is inactive until it reaches the digestive tract to be activated.
105
what is the most important mechanism for enzyme regulation throughout all species
reversible protein phosphorylation.
106
covalent modification of enzymes - what is protein phosphorylation
(the transfer of a phosphate group from ATP
onto the enzyme) is catalysed by a class of enzyme known as protein
kinases.
Phosphorylation of amino acids with an –OH
(serine, threonine, tyrosine) can change the shape of the active site and thus alter an enzyme’s biological catalytic activity.
Can change Vmax or Km.
107
what reverses protein phosphorylation
by a separate class of enzyme known as
protein phosphatases.
108
In mammals, the control of enzyme concentration can be achieved by control of what processes
Control of gene transcription
* Control of the stability of mRNA
* Control of protein synthesis (rate of mRNA translation)
* Control of the rate of protein degradation
109
- proteins are **positively charged at what pH**
one below their pI
110
- proteins are **negatively charged at what pH**
a pH above their pI
