Lecture 7: enzymes Flashcards
What happens during acid base catalysis?
The amino acid side chain donates or accepts protons to the substrate to form the product
What happens during covalent catalysis?
- The amino acid at the active site mounts a nucleophilic attack on the electrophilic site on the substrate
- This forms a transient covalent bond between the enzyme and the substrate, which lowers the activation energy
What happens during metal ion catalysis?
Metal ions act as cofactors for the enzyme
They can:
1. Bind to substrates and orientate it in an optimal spatial arrangement
2. Mediate oxidation reduction reactions
3. Stabilise or shield negative charges
What are the categories of enzyme cofactors?
- Prosthetic cofactors: tightly bound to the enzyme
- Coenzymes/co-substrates: loosely bound to the enzyme
What are the major classes of enzymes and what kind of reaction do they catalyse?
- Oxidoreductases: Redox reaction
- Transferases: move chemical groups
- Hydrolases: hydrolysis
- Lyases: non-hydrolytic bond cleavage
- Isomerases: intramolecular group transfer (isomerization)
- Ligases: Synthesis of new covalent bond between substrates
- Translocases: Catalyse the movement of ions or molecules across membranes
How to calculate rate of reaction?
v = (Vmax x [S]) / (Km + S)
What happens to the rate of reaction when the Michaelis constant is equal to the substrate concentration?
V = 0.5Vmax
What is Kcat?
- Turnover number
- Measures the maximum number of substrate molecules converted to product per unit time per active site
How do you calculate Kcat?
Kcat = Vmax / [E]T
What is the specificity constant and how do you calculate it?
- It measures the specificity of the enzyme for the substrate
- An overall measure of enzyme efficiency
- specificity constant = Kcat/Km
What do you get when you linearize a Michalis Menten plot?
Lineweaver-Burk plot
What is the lineweaver burk equation?
1/V = (Km/Vmax) / 1/[S] + 1/Vmax
How does a reversible competitive inhibitor work?
- The inhibitor is structurally similar to the substrate
- It competes with the substrate for binding to the active site
Are Vmax and Km affected by a competitive inhibitor?
Vmax no
Km increases (affinity decreases)
Can competitive inhibition be overcome?
Yes, by increasing substrate concentration
What is inhibitory constant?
- It is the concentration of the inhibitor required to occupy 50% of the enzyme’s active site
How does a non competitive inhibitor work?
- It binds to the allosteric site
- It can bind to the enzyme or the enzyme substrate complex to prevent the reaction from occuring
What happens to an enzyme’s Km and Vmax in the presence of a non-competitive inhibitor?
Km is unchanged
Vmax decreases
How do you calculate the observed Vmax in the presence of a non-competitive inhibitor?
Vmax (observed) = Vmax (original) / (1 + [I]/Ki)
How do you calculate the rate of reaction of an enzyme in the presence of a non-competitive inhibitor?
V = Vmax[S] / (Km + [S])(1 + [I]/Ki)
How does uncompetitive inhibition work?
- Inhibitor binds to ES complex
- It alters the way the ES complex would dissociate to release the product or the way the substrate binds to the enzyme
What happens to an enzyme’s Km and Vmax in the presence of a uncompetitive inhibitor?
Km decreases
Vmax decreases
Why does Vmax decrease when using a uncompetitive inhibitor?
When substrate concentration increases, more inhibitor molecules bind to the ES complex
Why does Vmax decrease when using a non-competitive inhibitor?
Inhibition cannot be overcome by increasing substrate concentration
Are irreversible inhibitors competitive or non competitive?
Can be both
What happens during irreversible inhibition?
- A covalent bond is formed between the inhibitor and the active/allosteric site of the enzyme
- Permanently stops the catalysis, the restoration of enzymatic activity can only be done by synthesizing new enzymes
What are the main clinical uses of enzymes?
- As therapeutics
- As key components in diagnostic/analytical assays
When is enzyme replacement therapy used?
Used on patients with defective or aberrant enzyme activity in metabolic pathways
What is K1?
The forward rate constant (formation of ES complex)
What is K2?
The rate constant of the breakdown of ES into enzyme and product
What is K-1?
The rate constant of the dissociation of ES into enzyme and substrate
How do you calculate Km using K2, K1 and K-1 values?
Km = (K2 + K-1) / K1
