Proteins - Lecture Twelve Flashcards
How can the activities of enzymes be measured and compared?
Significance of kM
Characterises one enzyme-substrate pair (if an enzyme can act on different substrates, it will have different KM values for each)
kM
Is the substrate concentration needed to reach half Vmax
Units of kM
Units of concentration
Low kM
High affinity
High kM
Low affinity
Physiological significance of kM
In the cell, for a particular enzyme-substrate interaction, [S] is often below the kM, this means rate will rise to accomodate more substrate, tending to maintain steady state
kM specificity
Km is specific for the interaction with the enzyme and particular substrate
Isozyme glucoksinase
Glucoksinase found in the liver differs from the hexokinase found in many other tissue
Turnover number, kcat
The number of substrate molecules converted to product, per enzyme, per unit of time, when E is saturated with substrate, this helps define the activity of one enzyme molecules
If Michaelisp-Menten Model fits, Kcat =
k2, therefore describes the ‘rate-limiting’ step
The most effective enzymes should have
A high Kcat and a low kM
Kcat
Ability to turnover a lot of substrate into product, per second
kM
To achieve near Vmax
Viscosity of water
Sets an absolute upper limit at ~109 s-1M-1
Enzymes with Kcat/kM above 108s-1M-1
Perfect catalysts
Inhibitor
A compound that binds to an enzyme and reduces its activity
Inhibitors important because
Natural inhibitors regulate metabolism
Many drugs, poisons & toxins are enzyme inhibitors
Used to study enzyme mechanisms
Used to study metabolic pathways
Irreversible inhibitor
Binds covalently to the enzyme. Once the enzymes dead, it won’t come back to ‘life’
Reversible inhibitor
Nnot covalently bound to the enzyme. The enzyme can be reactived later
Types of reversible inhibitor
Competitive and non-competitive (pure or mixed)
Irreversible inihibitor
Binds to the enzyme and permanently inactivates it and reacts with a specific amino acid side chain, usually in the active site, and forms a covalent bond
Addition of the bulky tosyl-L-phenylalanine methylketone to the histidine
Disables the catalytic triad and fills the active site, blocking substrate binding
Reversible inhibitors
Binds to the enzyme but can subsequently be released, leaving the enzyme in its original active condition
Competitive inhibitors
Competes with the substrate for binding in the active site as inhibitor and substrate cannot bind at the same time
Enalapril and Aliskiren
Lowers blood pressure
Statins
Lowers serum cholesterol
Protease inhibitors
AIDS drug
Juvenile hormone esterase
Pesticide target
Tamiflu
Inhibitor of influenza neuraminidase
Non-competitive inhibition
Inhibitors bind at a different site than the substrate, when they bind to the enzyme it alters the shape of the active site
In pure non-competitive inhibition, the binding of the inhibitor has no effect on the binding of the substrate
Non-competitive inhibitors
Are allosteric effectors
Pure non-competitive inhibition
Binding inhibitors changes the structure of the active site such as that the substrate still binds but the transition state stabilisation is no longer optimal, this results in the Vmax decreasing but the kM staying the same
Mixed non-competitive inhibition
Binding of the inhibitor does affect the binding of the substrate resulting in Vmax decreasing and kM increasing
“Binds substrate worse and is worse at turning over”
