ENZYMES lectures 1-3 Flashcards
1
Q
What is an enzyme (define)
A
A biological catalyst, which brings about one particular chemical reaction but itself remains unchanged
2
Q
what is an isoenzyme
A
Individual members of a set of enzymes, which catalyse the same reaction but differ in amino acid composition and have different properties
3
Q
what is the active site?
A
A region on an enzyme that binds to a protein or other substance during a reaction.
4
Q
what is an allosteric site
A
A regulatory site, located elsewhere to the active site where small molecules (other than cofactors and prosthetic groups) bind and effect a function change in the active site by causing a conformational change in the enzyme.
5
Q
What is a prosthetic group?
A
A complex in which a metal ion or small organic molecule is permanently bound to the apoenzyme by covalent bonds
6
Q
what are the parts of a prosthetic group called?
A
- Protein component known as apoenzyme, non protein component known as prosthetic group where
7
Q
what is a cofactor?
A
an organic molecule required to activate an enzyme, examples: coenzyme A (pantothenic acid), NAD (naiacin) many vitamins are converted to co-enzymes)
8
Q
what are the two fundamental properties of an enzyme
A
- They increase the reaction rate with no alteration of the enzyme
- They increase the reaction rate without altering equilibrium
9
Q
How do enzymes increase reaction rates?
A
- Enzymes reduce the activation energy —> decreasing the amount of energy required to form a complex of reactants that is competent to produce reaction products.
- complex is known as activated state or transition state complex for the reactions
↳ Lowers the amount of energy required to achieve the transition
state
10
Q
What rate do enzymes convert substrate to product?
A
Slow enzyme will convert 250 substrate molecules to products per second, fast one converts 600000/sec
11
Q
What is the Induced fit model?
A
- Binding of substrate causes conformation change in active site
- the initial interaction between enzyme and substrate is relatively
weak, but these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to be altered. - After binding, one or more mechanisms of catalysis generates transition state complexes and reaction products.
12
Q
Wha is the lock and key model?
A
- A negative impression of the substrate is considered to exist on the enzyme surface
- The substrate fits in this binding site just as a key fits into the lock
- Hydrogen and ionic bonding and hydrophobic interactions contribute in binding substrate to the binding site
- This model gives a rigid picture of the enzyme and cannot account for the effects of allosteric ligands
13
Q
what are the 4 mechanisms of catalysis
A
- bond strain
- proximity and orientation
- acid-base
- covalent
14
Q
what is Catalysis by bond strain
A
- induced fit
- substrate does not fit active site at first, changes conformation and shape
15
Q
what is Catalysis by proximity and orientation
A
- While free in solution, substrates tumble and collide.
- The probability for
collision at the angle necessary to change chemical interactions is low. - When bound to enzymes, two substrates can be oriented such that a reaction is more likely
- enzymes align reactive chemical groups and hold them close together
16
Q
What is catalysis by acid-base
A
- The acceleration of a reaction is achieved by catalytic transfer of a
proton. - This is the most common type of chemical catalysis.
- Acidic or basic side chains of AA’s in the active site transfers H+ to
or from the substrate, destabilising a covalent bond in a substrate
17
Q
What is covalent catalysis (example)
A
- Substrate forms a transient covalent bond with residues in the active site - reduces E of later transition states
- eg serine proteases such as Acetylcholinesterase
- Glutamate, histidine and serine (catalytic triad) in active site
- Histidine and glutamate donate electrons to the serine, making
- it reactive
- Forms a covalent bond with acetylcholine (AC)
Acetylcholine bond is broken down to acetate and choline
18
Q
what are the 3 ways enzymes are used in diagnosis of disease?
A
- Automated enzymatic assays
- immunochemical analysis
- measuring serum enzyme concentration
19
Q
What are automated enzymatic assays and the components needed?
A
- determination of substances in blood, plasma/serum and urine -
E-components of commercial kits:
- Determination of glucose - glucose oxidase, peroxidase - Cholesterol - cholesterol esterase, cholesterol
- Urea - urease
20
Q
what is immunochemical analysis?
A
- Elisa (=enzyme-linked immunosorbent assay)
- peroxidase, alkaline phosphatase
- Two main categories of ELISA: those designed to demonstrate
the presence of antibody and those designed to demonstrate
presence of antigen
21
Q
what is measuring serum enzyme concentrations?
A
- Examining enzyme activities in blood/serum can help ID the location & extent of disease:
- Enzymes specific to blood - high [E]
- Enzymes specific to tissues low [E] - i.e. no functional role in blood, leaked into the bloodstream due to injury
- clinical signs frequently non-specific
22
Q
What is enzyme kinetics?
A
the study of the binding affinities of substrates and inhibitors and the maximal catalytic rates that can be achieved
23
Q
what are regulatory enzymes
A
occupy key points in metabolic pathways and their activities are increased or decreased by specific mechanisms deigned to control the overall activate of the whole metabolic pathway
24
Q
what are ‘Slave’ /constitutive enzymes
A
the activities of these enzymes are controlled by their environment. As long as pH and temperature are acceptable and there are no inhibitors present, the activities are primarily determined by the supply of substrate
25
what is the effect of pH on enzyme kinetics
- Enzymes carry net molecular charges
- Operate within specific pH range and maximally at a specific pH
- Optimum varies but most enzymes work best at a pH around 7-9
26
what does the pH of surrounding medium do to enzymes?
affects:
- State of ionisation of aa’s in active site
- State of ionisation of substrate
- Nearly all enzymes ell shaped pH velocity profile
27
what is the effect of temperature on enzymes
- Increasing temperature increases rate of reaction
- Increases kinetic energy in the substrate and enzymes
- Optimum between 35-45 degrees Celsius
- Above optimum, heat denaturation of enzyme occurs
- Low temperatures can make enzymes reversibly inactive
- Freeze or refrigerate samples until testing can be done
28
What is Vmax
- maximum velocity att which an enzyme can function
- maximum velocity is reached when an enzyme is saturated with substrate (high substrate concentration)
29
why does Vmax depend on substrate conc
- Dependent upon enzyme concentration
- As substrate concentration increases, substrate is more likely to bump into the active site and more products are produced
- As substrate concentration goes up, velocity goes up
When there is so much substrate that all the enzymes are filled with substrate at all time, Vmax results
- At Vmax, increasing substrate no longer increases velocity
30
is Vmax the same for all substrates wth an enzyme?
no, determined for each substrate
- An enzyme will have as many Km values as it has substrates
31
is Vmax dependent on enzyme concentration?
yes
- Doubling amount of enzyme doubles the Vmax
32
what is Km?
- substrate concentration needed for half maximum velocity - measured in molarity (moles/L)
- Gives an indication of the affinity that the enzyme has for its substrates:
- High Km = weak binding between enzyme and substrate
- Low Km = strong binding between enzyme and substrate
33
is a larger or smaller Km better?
- Smallest Km value is better = tight binding
34
is Km dependent on enzyme concentration
- no, independent
35
how to calculate Vmax/Km (simple)
1. Graph initial velocity (Vo) vs Substrate concentration
2. estimate Vmax from peak (asymptote)
3. Calculate Vmax/2
4. go down to x axis from this point and find Km
36
How to calc Vmax/Km using lineweaver-burk plots
- plot 1/v (y) vs 1/substrate conc (x)
- 1/Km = x intercept
- 1/Vmax = y intercept
37
what is zero order kinetics?
Enzyme must be the only reactant that limits the reaction
38
what should the substrate concentration be during zero order kinetics to be considered excess
10-50 x Km
39
what is specific activity
the activity expressed relative to a fixed value such as weight of tissue or volume of fluid
40
what are the conditions needed for measuring the activity of an enzyme
1. Substrate concentration - non limiting
2. Enzyme concentration - limiting
3. Co-factor concentration - non limiting
4. Incubation time - relatively short
41
when measuring the substrate concentration using an enzyme, what conditions should apply?
1. Substrate concentration - limiting
2. Enzyme concentration - non limiting
3. Co-factor concentration - non limiting
4. Incubation time - relatively long
42
What are the 2 broad classes of enzyme inhibitors?
1. Irreversible
2. Reversible
43
why are some inhiibitors irreversible and give some examples
- Covalently and permanently bind to enzyme
- Cannot be reversed
- Usually poisons - cyanide, carbon monoxide and
polychlorinated biphenols (PCBs), sarine gas, organophosphates
- Penicillin - inhibition of bacterial cell wall synthesis
44
why are some inhibitors reversible
Inactivates enzyme through enzyme non covalent, more easily reverse, transient interactions
45
what are the 3 types of reversible inhibitors
- Competitive
- Non competitive
- Uncompetitive (rare)
46
what is competitive inhibition
- competes with substrate
- Resembles the normal substrate and competes with it for the active site
- Inhibitor is reversible by substrate, add more substrate to compete with the inhibitor
- Add more substrate to flush out inhibitor
47
what is non-competitive inhibition
- can bind to enzyme with or without enzyme substrate
- Non-competitive inhibitor can bind to enzyme before or after substrate
- Inhibition cannot be reversed by substrate
48
what is uncompetitive inhibition
- only binds to enzyme-substrate complex (allosteric site)
- Uncompetitive inhibitor cannot bind before the substrate
- Inhibition cannot be reversed by substrate
49
effect of competetive inhibition on Km and Vmax
Km = higher
Vmax = same
50
effect of non-competetive inhibition on Km and Vmax
Km = same
Vmax = lower
51
effect of uncompetetive inhibition on Km and Vmax
Km = lower (substrate taken up by both ES and EIS complexes so appears as higher affinity)
Vmax = lower
52
explain the inhibition occurring in vitamin K rodenticide poisoning:
- competitive inhibition of vitamin K epoxide reductase -> rodenticides are vitamin K antagonists (block)
- vitamin K is an essential cofactor for carboxylase in synthesis of prothrombin (factor II)
- rodenticides inhibit vitamin K epoxide reductase, resulting in inability of body to recycle vitamin K = Vitamin K deficiency
- therefore viitamin K cannot act as co factor to form active clotting factors from inactive ones beside carboxylase
53
treatment for rodenticides
supplying exogenous vitamin K to animal/human
54
what are organophosphates (OP)
derivatives of phosphoric or phosphoric acid
55
what do OP's do to cause illness?
- OPs affect serine proteases (eg acetylcholinesterase -
ACE) in the same way that sarin gas does.
- Serine proteases have a reactive serine in their active site because OPs bind specifically with reactive series to form an inactive complex which is stable indefinitely -> causes nerve impulses to be permanently transmitted
- if ACE inhibited by OP = continued depolarisation and muscle contraction
56
what ways are enzymes regulated by regulatory enzymes
- Allosteric regulation
- Reversible covalent modification
- Proenzyme activation (proteolytic cleavage)
- Rate of enzyme synthesis and degradation and compartmentation
- Stimulation and inhibition by control proteins such as calmodulin
57
how do regulatory enzymes alter enzyme activity using allosteric modification?
- effector molecules bind to enzyme at allosteric sites, well removed from the catalytic site
bring about catalytic modification by binding to the enzyme at distinct allosteric sites, causing conformational changes that are transmitted through the bulk of the
- Allosteric modification of regulatory enzyme activity
protein to the catalytically active sites.
- causes conformational change (inactive to active form)
58
what is reversible covalent modification
- covalent attachment of another molecule to modify enzyme activity
- usually addition or removal of phosphate, methyl or acetyle
- usually reversible and rapid modifications
59
what is a kinase and phosphatase
kinase -> adds a phosphate group
phosphatase -> removes phosphate groups
60
What is proenzyme activation (proteolytic cleavage)
- more rapid increase in enzyme activity
- non-reversible
- cleavage of something will produce an active enzyme from an inactive enzyme (zymogens/proenzyme)
- can be stored for long time as zymogen
61
Explain Rate of enzyme synthesis/degradation and compartmentalisation
- many rate-limiting enzymes are present in very low concentrations and have very short half-lives and therefore the levels of the enzyme can be altered by increased synthesis
- this is a slow mechanism for regulating enzyme concentration
- compartmentalisation is sequestering enzymes in
compartments where access to their substrates is limited
- Generally anabolic (synthetic) and catabolic (breakdown) pathways are separated into different compartments within the cell
62
what is regulation by control proteins?
- the effector molecules are proteins, which need to be modified in some way before they are effective at regulating enzyme activity
- eg. Calmodulin = calcium-modulating protein
- Mediates many calcium driven metabolic reactions
- 4 binding sites for calcium
When calcium increases, calmodulin binds calcium - Calmodulin is activated
- Regulates other proteins
Calcium dependent allosteric activator once is has been activated by calcium
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