Introduction to Enzymes Flashcards
How do enzymes work?
Act as biological catalysts. They speed up the rate at which equilibrium is reached; they do not shift the position of the equilibrium. They do this by decreasing the activation energy. Enzymes provide catalytically component groups for a specific reaction mechanism through residues on the active site.
- Bind to the substrates in the correct orientation that is optimised for the reaction.
- By binding, enzymes stabilise the transition state of the reaction. The reaction therefore acts quicker.
What are the key features of the active site of enzymes?
The active site is a 3D space comprising of crucial amino acids in the correct orientation that is optimised for the reaction. The substrate binds to the active site using weak interactions. The residues may not near each other in the primary sequence, but come together when the tertiary structure is formed. Often the active site is a very small part of the entire structure. Due to unique arrangement of the active site, the enzyme is usually specific to only a few amino acids.
How is enzyme activity measured?
Using the Michealis-Menton Model.
km = k2 + k3/k1 where k1 is the rate of formation of the enzyme-substrate complex. k2 and k3 are the rate constants of dissociation of the enzyme substrate complex (k2 is the dissociation to enzyme and substrate and k3 the dissociation to produce the product). This model assumes k3 is much smaller than k2 (it is the rate determining step). We measure activity by measuring the rate at which product is produced or substrate is lost.
Define km and Vmax
km is the concentration at which the enzyme has reached half of Vmax. It is essentially a measure of the enzymes affinity for substrate.
Vmax is the maximum rate of the enzyme. At this point all of the enzymes would be saturated. This assumes that there is an infinite number of substrate molecules.
What is the Michealis-Menton Equation?
V = Vmax [S]/Km + [S]
It can also be represented as a Lineweaver-Burk double reciprocal plot as:
1/V = km/Vmax(1/[S]) + 1/Vmax
What are the factors affecting the rate of enzyme reaction?
Temperature: Increasing the temperature, increases the rate of reaction. Past the optimum temperature rate decreases. At extremely high temperatures the protein will denature - weak interactions break and the enzyme unwinds. Most of the enzymes in our body have evolved to function optimally at physiological temperatures.
pH: Most have an optimum pH equal to physiological pH. At extreme pH (low and high) the proteins will denature and enzyme rate decreases.
Inhibitors.
How do irreversible inhibitors affect enzymes? Give examples
Irreversible inhibitors usually covalently modify the enzymes. This is affects the side chains of enzymes in the active site (some irreversible inhibitors can dissociate with the enzyme producing an inactive form of the enzyme). An example of irreversible inhibitors is AChE inhibitors in nerve agent such as Sarin. Here there is covalent modifications of Serine. Another example is carboxymethylation os cysteine side chains in iodoacetamine.
What is the method of action of competitive reversible inhibitors?
Either the substrate or the inhibitor can bind - but cannot bind simultaneously. In many cases, this involves both competing for the active site. This is usually a non-covalent interaction. Inhibition can be overcome by increasing substrate concentration. The value of Vmax is the same (as the substrate will eventually outcompete the inhibitor) but Km is changed.
What is the method of action of a non-competitive inhibition?
The inhibitor and the substrate can bind simultaneously to the enzyme at independent sites. The inhibitor alters the conformation and accessibility of the active site, The reaction cannot occur - the substrate may still be able to bind but the catalytically component groups are nit in the correct place for the reaction to occur. As a result increasing substrate concentration will not overcome the inhibition. As a result Vmax changes but Km is the same.
Give an example of competitive reversible inhibition.
Methotrexate. Inhibits dihydrofolate reductatase. The drug is very similar to dihydrofolate but cannot be processed by the enzyme. It is a drug used for inflammation at low doses and in cancer treatment in high does.
Aspirin inhibits cyclo-ooygenases. Cyclo-oxygenases catalyses the synthesis of prostaglandins using lipids as a substrate (arachidonic acid). Aspirin causes a covalent modification - it is non-reversible. Inhibitor binding in Ibropofen is also competitive but is reversible.
