Factors Affecting Enzyme Activity

Enzymes are large globular proteins

They have a precise 3-D shape Some have quaternary structure The active site (blue) represents a tiny part of the molecule

RuBisCo

A reminder about protein structure

Protein structure is achieved by the precise folding of secondary structures to form a tertiary structure held together by a range of bond types between Rgroups (or side-chains)

Amylase

Some reaction kinetics

Some reaction kinetics

The Lock and Key analogy

The Lock and Key analogy

Induced fit

Enzymes and temperature: a tale of two effects

Reaction rate / arbitrary units

Collision rate of enzymes and substrates Number of enzymes remaining undenatured

Temperature / oC

Enzymes and temperature

Reaction rate / arbitrary units

Increasing kinetic energy increases successful collision rate

Temperature / oC

Enzymes and temperature

Reaction rate / arbitrary units

Permanent disruption of tertiary structure leads to loss of active site shape, loss of binding efficiency and activity

Temperature / oC

Enzymes and temperature

Optimum temperature
Reaction rate / arbitrary units

Temperature / oC

Enzymes and pH
The precise shape of an enzyme (and hence its active site) depends on the tertiary structure of the protein Tertiary structure is held together by weak bonds (including hydrogen bonds) between R-groups (or side-chains) Changing pH can cause these side chains to ionise resulting in the loss of H-bonding

Enzymes and pH
Optimum pH
Reaction rate / arbitrary units

Either side of the optimum pH, the gradual ionising of the side-chains (R-groups) results in loss of Hbonding, 3o structure, active site shape loss of binding efficiency and eventually enzyme activity

pH

Enzymes and pH
Optimum pH
Reaction rate / arbitrary units

This loss of activity is only truly denaturation at extreme pH since between optimum and these extremes, the loss of activity is reversible

pH

Enzymes and pH

Enzymes and [S]

As soon as a reaction begins, [S] begins to fall and so it is important that initial reaction rates are measured

Initial reaction rate / arbitrary units

[S]

Initial reaction rate / arbitrary units

Enzymes and [S]

[S]

Enzymes and [S]

Increasing [S] increases collision rate and increases reaction rate

Initial reaction rate / arbitrary units

[S]

Enzymes and [S]

All active sites are occupied. Enzymes are working at maximum rate.

Initial reaction rate / arbitrary units

All active sites are not occupied

[S]

Enzymes and [S]

Maximum turnover number or Vmax has been reached

Initial reaction rate / arbitrary units

[S]

Enzymes and [enzyme]

Can we explain this in terms of the proportions of active sites occupied?

Initial reaction rate / arbitrary units

What factor is limiting here?

[Enzyme]

Enzymes and inhibitors

Inhibitors are molecules that prevent enzymes reaching their maximum turnover numbers Some inhibitors compete with the substrate Active site directed inhibition for the active site Some inhibitors affect the active site shape Non-active site directed elsewhere by binding to the enzyme inhibition on the enzyme

Active site directed inhibition

Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate

Inhibitor Enzyme

Active site directed inhibition

Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate

Enzyme activity is lost

Enzyme/Inhibitor complex

Enzymes and active site directed inhibition

At low [S], the enzyme is more likely to bind to the inhibitor and so activity is markedly reduced Initial reaction rate / arbitrary units

Uninhibited Inhibited
[S]

Enzymes and active site directed inhibition

As [S] rises, the enzyme is increasingly likely to bind to the substrate and so activity increases Initial reaction rate / arbitrary units

Uninhibited Inhibited
[S]

Enzymes and active site directed inhibition

At high [S], the enzyme is very unlikely to bind to the inhibitor and so maximum turnover is achieved Initial reaction rate / arbitrary units

Uninhibited Inhibited
[S]

 Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate

Non-active site directed inhibition

Inhibitor Enzyme

 Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate

Non-active site directed inhibition

Enzyme

Active site is changed irreversibility

 Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate

Non-active site directed inhibition

Enzyme

Activity is permanently lost

Enzymes and non-active site directed inhibition

Can we explain this graph in terms of limiting factors in the parts of the graph A and B?

Initial reaction rate / arbitrary units

[S]