Enzymes

Question 1

How do enzymes catalyse reactions?

Answer 1

1. They accelerate the movement towards equilibrium.
2. Lower the activation energy of biochemical reactions.
   - entropy reduction
   - desolvation; weak bonds replace H bonds between substrate and aqueous solution.
   - induced fit; conformational changes
3. Increases rate of spontaneous reactions.

Question 2

How might an enzyme catalysed reaction change?

Answer 2

More substrate will result in a higher initial rate of reaction. The reaction will slow down as the availability of active sites reduces.
An increase in temperature will increase the rate.
Changes in pH will alter the charge of the active site.
A decrease in the enzyme concentration will decrease the rate.
Any extreme changes will denature the enzyme.

Question 3

Michaelis constant

Answer 3

the concentration of a given substrate which catalyses the associated reaction at half the maximum rate.

Vo=initial velocity, Vmax=mximum reaction velocity, Km=michaelis constant.
Km=1/2Vmax[S]

Km can be calculated from a hyperbolic reaction curve or from a Lineweaver-Burk plot (more accurate).

Km gives you an idea of the affinity an enzyme has for its substrate. A large Km=less stable ES=low affinity. A small Km=more stable ES=high affinity.

Question 4

Competitive inhibition

Answer 4

Something competes with the substrate for the active site. It increases Km and is reversible by increasing the number of available active sites. Vmax remains unchanged.

Question 5

Non-competitive inhibition

Answer 5

Something binds to the enzyme altering the active site, preventing the substrate from binding. Km is unchanged but Vmax decreases as the enzyme can’t work quick enough.

Question 6

Uncompetitive inhibition

Answer 6

This is irreversible. Something binds to the enzyme after the substrate has bound so locks the substate in place and prevents the reaction occuring.

Question 7

Allosterically-regulateted enzymes

Answer 7

Allosteric effectors are usually cell metabolites that bind non-covalently to a site other than the active site.

Concerted model; each substrate exists in 2 states; tense and relaxed. One binds substrate well but with no substrate the enzyme flips between the 2 states. All subunits must be in the same conformation so when one substrate binds it locks all binding sites. Allosteric activators stabilise the open conformation whilst inhibitors close it.

Sequential model; there is no flipping between open and closed. The substrate binding causes a change in one sub-unit causing a change in another and so on.

Question 8

Covently-modified enzymes

Answer 8

Phosphorylation; approx 30% of all eukaryotic proteins. Kinases add a phosphoryl group and phosphotases remove it. It can occur at a single site, multiple sites or multiple phosphorylations at one site. Multiple phosphorylation sites allow very fine control of enzyme function. The enzyme doesn’t exist in an on/off state but has finely tuned activity dependant on the signals it recieves.

Proteolytic cleavage; enzymes can exist as an inactive precursor protein (proprotein/proenzyme). Proproteins can be cleaved to give active protein by proteases. Eg digestive enzymes.

Question 9

Discuss the use of enzyme assays in clinical diagnosis.

Answer 9

Enzymes in the wrong place indicate disease. Enzyme activity can be measured and compared with normal. We can seperate different forms of enzymes by electrophoresis and examine the pattern. This is useful to distinguish between isoenzymes (multiple enzymes that catalyse the same reaction).