Enzyme Kinetics Flashcards
What are the principles of column chromatography?
This method of protein purification takes advantage of differences in protein charge, size, binding sites and other properties. A porous solid material with appropriate chemical properties is held in a column, and a buffered solution migrates through it.
Most enzymes have a rate enhancement between what?
All enzymes are what?
Give examples of cofactors? What do cofactors do?
Give an example of a cofactor?
10^5 - 10^17
All enzymes are proteins except for a catalytic group of RNA molecules.
Fe2+, Mg2+, Mn2+ and Zn2+ are cofactors. They are required for the enzyme to catalyse molecules properly. And example is the Zn2+ cofactor in carbonic anhydrase.
What is size exclusion chromatography?
This method separates enzymes by their size. The column contains porous beads that allow smaller enzymes inside. This means larger enzymes are removed quickly in earlier elutions.
What is ion exchange chromatography?
In this method proteins are added to the column containing cation exchangers. The proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers proteins with a net negative charge move through the column and elute quicker.
What is affinity chromatography?
The beads within the column have covalently ligands. If the proteins added have an affinity for the ligands its progress through the column is slowed down.
Describe what would happen to the concentration of product when an enzyme is added to a substrate.
The concentration would increase quickly at first but would eventually plateau as the substrate would be used up. When the concentration is 100% it would have completely plateaued.
Once the elutions have been separated using a technique how can the enzyme of interest be found?
What is the equation for enzyme activity?
Can be found by finding the initial velocity of each fraction, the one with the highest would be the enzyme wanted.
Specific activity = moles of substrate converted per unit time per mass of protein
Units are moles min^-1
If the rate of a enzyme catalysed reaction is greater in the absence of molecule X than in the presence of molecule X, what is molecule X classed as?
List the production pathway compounds for prostaglandins, and name the enzyme involved converting the intermediate to the product.
An inhibitor
Phospholipid -> arachidonic acid -> prostaglandins
The enzyme converting arachidonic acid to prostaglandins is cyclooxygenase.
Name an inhibitor of cyclooxygenase. What type of inhibitor is it and describe what that means.
Flurbiprofen
It is a competitive inhibitor which means it competes with the substrate for a place in the enzyme’s active site. While the inhibitor is in the active site it prevents the binding of the substrate.
Competitive inhibitors have to be what in relation to the substrate.
How does the inhibition of cyclooxygenase give an insight into catalysis?
They have to be structurally similar to the substrate.
Both flurbiprofen and ibuprofen are inhibitors of cyclooxygenase, a characteristic of both is that they share a benzene ring. Arachidonic acid does not possess a benzene ring which suggests that that is not the form it takes in the enzyme.
Describe how uncompetitive inhibitors work.
They only bind to enzyme-substrate complexes and prolong the time take for the product to leave the enzyme. This reduces both the Km and Vmax.
Describe how mixed inhibitors work.
These bind to either enzymes or enzyme-substrate complexes. They have the same effect as uncompetitive inhibitors in that they lower the Km and Vmax.
What is the enzyme that catalyses the conversion of glycogen to glucose-1-phosphate?
Glycogen phosphorylase
