BB 9 10 11 Enyme Kinetics Flashcards
(56 cards)
0
Q
Trypsin
A
cleaves only after arginine and lysine residues
1
Q
Enzymes
A
- biological catalysts
- accelerates rate of reactions
- function by stabilizing transition states in reactions
- don’t change equilibrium of reactions
2
Q
Thrombin
A
cleaves between arginine and glycine in particular sequences
3
Q
Papain
A
cleaves all peptide bonds irrespective of sequence
4
Q
DNA Polymerase I
A
adds nucleotides in sequence determined by template strand
5
Q
Cofactors
A
small molecules essential for enzyme catalysis
6
Q
Apoenzyme
A
enzyme without its cofactor
7
Q
Holoenzyme
A
enzyme with its cofactor
8
Q
Free energy
A
- the difference between its reactants and its products
* independent of reaction path
9
Q
Negative delta G
A
- reaction may occur spontaneously, doesn’t mean it will
* exergonic
10
Q
Positive delta G
A
- doesn’t occur spontaneously
- requires energy input
- endogonic
11
Q
Delta G
A
- energy of the endpoints
* tells nothing about rate of reaction
12
Q
Equilibrium constant – Keq
A
defines rate of reaction
13
Q
Activation energy
A
- reactions go via a high energy intermediate
- reduces the rate at which equilibrium is reached
- larger activation energy = slower rate of reaction
14
Q
Transition state theory
A
- enzymes reduces the activation barrier
- transition state energy becomes smaller
- need to put energy in even though end up releasing energy
15
Q
Active site
A
- region that binds the substrate
- a 3D structure formed by groups that can come from distant residues in the enzyme (tertiary structure)
- take up a small volume of the enzyme
- unique chemical environments, usually formed from a cleft or crevice in the enzyme
16
Q
Active sites often exclude
A
- water
- non-polar, enhances binding of substrates, allow polar catalytic groups to acquire special properties required for catalysis
17
Q
Active sites bind substrates with
A
- weak interactions
* eg electrostatic, hydrogen bonds, Van der Waals, hydrophobic interactions
18
Q
The specificity of an enzyme for its substrate(s) is critically dependent on
A
• the arrangement of amino acid residues at the active site
tertiary structure
19
Q
Catalytic specificity depends on
A
- binding specificity
* activity of enzymes regulated at this stage
20
Q
Evidence for ES complexes
A
- saturation effect
- crystallography (structural data)
- spectroscopic data
21
Q
Saturation effect
A
• at constant enzyme concentration, reaction rate increases with substrate until Vmax is reached
22
Q
Catalytic groups
A
- amino acid side chains in the active site associated with the making and/or breaking of chemical bonds
- make up the active site
23
Q
Induced fit model
A
- Koschland, 1958
- substrates and enzymes are flexible and dynamic
- the enzyme changes shape in order to optimise its fit to the substrate only AFTER the substrate has bound
24
First order reaction
| uni-molecular
V = k [A]
| k: s-1
25
Second order reaction
| bi-molecular
v = k [A][B]
| k: M-1 s-1
26
Michaelis-Menten Model
* describes the kinetic property of enzymes
* at a fixed concentration of enzyme, increasing substrate concentration increases reaction rate
* maximal reaction velocity w/ saturating substrate for a fixed amount of enzyme implies a specific ES complex is a necessary intermediate in enzyme catalysis
* add more substrate to point where it has no effect
27
Michaelis-Menten Equation
* relates the rate of catalysis to the concentration of the substrate
* plotting initial velocity of a reaction against substrate concentration produces the Michaelis-Menten curve
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Km for any enzyme depends on
* pH
* temperature
* ionic strength
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Km
* substrate concentration required for the reaction velocity to be half the maximal value
* Michaelis-Menten constant
* tells us enzyme-substrate affinity
30
Vmax
* maximal velocity of the reaction
* rate/velocity at which all enzyme active sites are filled
* number of substrate molecules converted into product by an enzyme molecule per unit time when enzyme is fully saturated
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Catalytic power
* enzyme’s turnover
* maximum number of substrate molecules converted into product by an enzyme molecule un unit time
* E fully saturated, equal to kinetic constant k2 = AKA kcat
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Diffusion limit
the max value of k2/Km
33
The perfect enzyme is limited by
diffusion
• k+1 = how often the enzyme collides with its substrate
• only limited by the rate of collisions = diffusion limited
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Multiple substrate reactions can be classified into classes:
* sequential reactions
| * double displacement (ping-pong) reactions
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Sequential reactions
* all substrates bind to enzyme, forming ternary complex
| * can be ordered or random interactions
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Double-displacement reactions
* AKA ping-pong reactions
| * one or more products released before all substrates bind to enzyme
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Allosteric Enzymes consist of
* multiple subunits
* multiple active sites
* sigmoidal
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Inhibitors
molecules that prevent enzymes from working
• may regulate enzymes
• can act as medicinal drugs or toxins
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2 main types of enzyme inhibition
* irreversible
| * reversible
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Irreversible inhibition
the inhibitor is tightly bound to the enzyme (sometimes covalently)
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Reversible inhibition
inhibitor can bind and dissociate from the enzyme
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Competitive inhibitors
* bind to the active site of enzymes
| * reduce the effective substrate concentration
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Non-competitive inhibitors
* stop the enzyme from working by changing the conformation of the active site
* reduce the effective enzyme concentration
* don’t bind to active site
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Uncompetitive inhibitors
* bind to the ES complex
| * cannot be overcome by adding more substrate
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Methotrexate
* reversible inhibitor
* structural analog of substrate for DHFR
* prevents nucleotide synthesis
* used to treat cancer
* biosynthesis of purines and pyrimidines
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Penicillin
• irreversible inhibitor
• covalently modifies transpeptidase
• inhibits bacterial cell wall synthesis, killing bacteria
(peptidoglycan)
• reacts with serine residue in active site
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Competitive inhibition
* Km increases
| * Vmax stays the same
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Non-competitive inhibition
* Vmax lowered
| * No change in Km
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Uncompetitive inhibition
* binding to ES stops reaction
| * both Vmax and Km are lowered
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Whether Km or Vmax change depends on
where the inhibitor binds to the enzyme (type of inhibition)
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Transition state analogs
* mimic transition state = effective inhibitors
* enzymes work by stabilizing transition state
* TS analogs bind tightly to the active site
* very good COMPETITIVE inhibitors
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Catalytic antibodies
* stabilize transition state = catalyze reaction
| * antibodies which recognize a transition state function as catalysts (bind and stabilize)
53
Enzyme regulation can be
* cooperative
| * allosteric
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Cooperative regulation
• binding of substrate to one binding site helps binding to other active sites
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Allosteric regulation
* involves product inhibition
* product regulates work of first enzyme in pathway
* used to control flux through metabolic pathways
* feedback (negative) inhibition
* DON’T SHOW MICHAELIS-MENTEN KINETICS (multiple subunits and multiple active sites = sigmoidal)
