BB 9 10 11 Enyme Kinetics Flashcards
Trypsin
cleaves only after arginine and lysine residues
Enzymes
- biological catalysts
- accelerates rate of reactions
- function by stabilizing transition states in reactions
- don’t change equilibrium of reactions
Thrombin
cleaves between arginine and glycine in particular sequences
Papain
cleaves all peptide bonds irrespective of sequence
DNA Polymerase I
adds nucleotides in sequence determined by template strand
Cofactors
small molecules essential for enzyme catalysis
Apoenzyme
enzyme without its cofactor
Holoenzyme
enzyme with its cofactor
Free energy
- the difference between its reactants and its products
* independent of reaction path
Negative delta G
- reaction may occur spontaneously, doesn’t mean it will
* exergonic
Positive delta G
- doesn’t occur spontaneously
- requires energy input
- endogonic
Delta G
- energy of the endpoints
* tells nothing about rate of reaction
Equilibrium constant – Keq
defines rate of reaction
Activation energy
- reactions go via a high energy intermediate
- reduces the rate at which equilibrium is reached
- larger activation energy = slower rate of reaction
Transition state theory
- enzymes reduces the activation barrier
- transition state energy becomes smaller
- need to put energy in even though end up releasing energy
Active site
- 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
Active sites often exclude
- water
- non-polar, enhances binding of substrates, allow polar catalytic groups to acquire special properties required for catalysis
Active sites bind substrates with
- weak interactions
* eg electrostatic, hydrogen bonds, Van der Waals, hydrophobic interactions
The specificity of an enzyme for its substrate(s) is critically dependent on
• the arrangement of amino acid residues at the active site
tertiary structure
Catalytic specificity depends on
- binding specificity
* activity of enzymes regulated at this stage
Evidence for ES complexes
- saturation effect
- crystallography (structural data)
- spectroscopic data
Saturation effect
• at constant enzyme concentration, reaction rate increases with substrate until Vmax is reached
Catalytic groups
- amino acid side chains in the active site associated with the making and/or breaking of chemical bonds
- make up the active site
Induced fit model
- 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
First order reaction
uni-molecular
V = k [A]
k: s-1
Second order reaction
bi-molecular
v = k [A][B]
k: M-1 s-1
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
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
Km for any enzyme depends on
- pH
- temperature
- ionic strength
Km
- substrate concentration required for the reaction velocity to be half the maximal value
- Michaelis-Menten constant
- tells us enzyme-substrate affinity
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
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
Diffusion limit
the max value of k2/Km
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
Multiple substrate reactions can be classified into classes:
- sequential reactions
* double displacement (ping-pong) reactions
Sequential reactions
- all substrates bind to enzyme, forming ternary complex
* can be ordered or random interactions
Double-displacement reactions
- AKA ping-pong reactions
* one or more products released before all substrates bind to enzyme
Allosteric Enzymes consist of
- multiple subunits
- multiple active sites
- sigmoidal
Inhibitors
molecules that prevent enzymes from working
• may regulate enzymes
• can act as medicinal drugs or toxins
2 main types of enzyme inhibition
- irreversible
* reversible
Irreversible inhibition
the inhibitor is tightly bound to the enzyme (sometimes covalently)
Reversible inhibition
inhibitor can bind and dissociate from the enzyme
Competitive inhibitors
- bind to the active site of enzymes
* reduce the effective substrate concentration
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
Uncompetitive inhibitors
- bind to the ES complex
* cannot be overcome by adding more substrate
Methotrexate
- reversible inhibitor
- structural analog of substrate for DHFR
- prevents nucleotide synthesis
- used to treat cancer
- biosynthesis of purines and pyrimidines
Penicillin
• irreversible inhibitor
• covalently modifies transpeptidase
• inhibits bacterial cell wall synthesis, killing bacteria
(peptidoglycan)
• reacts with serine residue in active site
Competitive inhibition
- Km increases
* Vmax stays the same
Non-competitive inhibition
- Vmax lowered
* No change in Km
Uncompetitive inhibition
- binding to ES stops reaction
* both Vmax and Km are lowered
Whether Km or Vmax change depends on
where the inhibitor binds to the enzyme (type of inhibition)
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
Catalytic antibodies
- stabilize transition state = catalyze reaction
* antibodies which recognize a transition state function as catalysts (bind and stabilize)
Enzyme regulation can be
- cooperative
* allosteric
Cooperative regulation
• binding of substrate to one binding site helps binding to other active sites
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)
