Enzymes

Review

Learning Outcomes

• (h) Explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation energy and enzyme specificity.

• (i) Follow the time course of an enzyme-catalysed reaction by measuring rates of formation of products (e.g. using catalase) or rate of disappearance of substrate (e.g. using amylase).

• (j) Investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzyme catalysed reactions, and explain these effects.

• (k) Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity.

Learning Outcomes

• (h) Explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation energy and enzyme specificity.

• (i) Follow the time course of an enzyme-catalysed reaction by measuring rates of formation of products (e.g. using catalase) or rate of disappearance of substrate (e.g. using amylase).

• (j) Investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzyme catalysed reactions, and explain these effects.

• (k) Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity.

HIV protease

Mode of action of enzymes

• Enzymes speed up the rate of reaction

• By lowering the activation energy of the reaction

How does an enzyme reduce activation energy?

• When the enzymes and substrates are in close proximity in the correct orientation, they form an enzyme-substrate complex

• Binding aa residues hold the substrate in place with weak non-covalent bonds

Mode of action of enzymes

• This causes a strain on the bonds in the substrates

• The unstable transition state configuration is attained– Enzyme active site provides a

microenvironment that favours the reaction

• Since the transition state is less unstable than without the enzyme, the activation energy is reduced!

Mode of action of enzymes

• The catalytic aa residues’ R groups facilitate the reaction, making or breaking bonds in the substrate

• The product is formed and released from the active site.

Induced Fit hypothesis

• Active site is flexible in conformation (not precisely complementary to substrate)

• When substrate enters active site, active site conformation changes– Catalytic amino acid residues moulded into a

precise conformation and position close to chemical bonds in substrate

– Strains bonds in substrate– Lowers activation energy of reaction

Specificity

• Substrate specific• Conformation of active

site, as well as properties and positions of chemical groups in R groups, ensures that only complementary substrates will enter active site

• Explained by lock and key hypothesis

• Group specific• One enzyme can catalyze

reactions for a variety of substrates with similar structural or chemical properties

• Explained by induced-fit hypothesis

Learning Outcomes

• (h) Explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation energy and enzyme specificity.

• (i) Follow the time course of an enzyme-catalysed reaction by measuring rates of formation of products (e.g. using catalase) or rate of disappearance of substrate (e.g. using amylase).

• (j) Investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzyme catalysed reactions, and explain these effects.

• (k) Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity.

Time course of an enzyme catalysed reaction

• Analysed by looking at the rate of product formation or the rate of substrate disappearance

Time course…

• Initially, substrate concentration is high. Frequency of effective collisions between enzyme and substrate is high, leading to high rate of formation of products. Hence high rate of reaction (steep initial gradient)

• As reaction progresses, rate slows down and plateaus. This is because substrate concentration falls. Frequency of effective collisions falls, leading to lower rate of formation of products. When all substrate is used, no more product is formed (graph plateaus)

Catalase

• Decomposes hydrogen peroxide to water and oxygen

• Can measure rate of product (oxygen) formation as the gas comes out of the solution and can be collected in a gas syringe/inverted measuring cylinder

• Plot graph of volume of gas produced over time to show time course of reaction

• Rate of product formation at different time points of one experiment would be the gradient of the graph at those times

Amylase

• Hydrolyses amylose to glucose• Can measure rate of substrate breakdown

by testing how long it takes for a starch test on the reacting sample to become negative, i.e., no more starch

• The longer it takes for starch test to give negative result, the lower the rate of hydrolysis overall rate = 1/t (where t is the time taken to reach the achromic point

So where do the ‘rate against…’ graphs come from?

• If you want to plot a graph of rate of reaction (dependent variable) over enzyme concentrate (independent variable…

• Carry out one reaction at a certain enzyme concentration and plot the time course. Find the initial rate of reaction

• Do it again for another enzyme concentration and find the rate

• Do it again… and again… and again…• Until you get enough manipulated data points (of initial

rate) for the graph! Then you can see how rate changes as [enzyme]

changes

Learning Outcomes

• (h) Explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation energy and enzyme specificity.

• (i) Follow the time course of an enzyme-catalysed reaction by measuring rates of formation of products (e.g. using catalase) or rate of disappearance of substrate (e.g. using amylase).

• (j) Investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzyme catalysed reactions, and explain these effects.

• (k) Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity.

Enzyme concentration

Substrate concentration

Vmax and Km

Temperature

pH

Learning Outcomes

• (h) Explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation energy and enzyme specificity.

• (i) Follow the time course of an enzyme-catalysed reaction by measuring rates of formation of products (e.g. using catalase) or rate of disappearance of substrate (e.g. using amylase).

• (j) Investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzyme catalysed reactions, and explain these effects.

• (k) Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity.

Competitive inhibition vs Non-competitive inhibition

Competitive Non-competitive

Inhibitor structurally resembles substrate

Inhibitor does not resemble substrate

Inhibitor binds to active site of enzyme

Inhibitor binds to site other than active site

Maintains VmaxDecreases Vmax (effect is that of decreased [E])

Increases Km (like reducing affinity of enzyme for substrate)

Maintains Km (does not affect affinity of enzyme for substrate

Graphs for inhibition

Allosteric regulation

• Allosteric regulators bind to a specific site called the allosteric site on the enzyme (not the active site)

• Enzymes having allosteric regulation are usually multimeric (more than one polypeptide subunit)

• They oscillate between active and inactive conformations

• Binding of an activator stabilizes the active form• Binding of an inhibitor stabilizes the inactive form