Chap 10 allosteric10.1

Why regulate enzyme activity?

Regulation allows for efficient use of resources by the cell.

Metabolic pathways rarely stand alone, and usually intersect with numerous other pathways

In enzyme pathways of several steps there are often cataslyzed by key enzymes that are rate-limiting in controlling the flux through the pathway

This integrating expression of the pathway with other metabolic needs of the cell

Why regulate enzyme activity?

Regulation allows for efficient use of resources by the cell.

Metabolic pathways rarely stand alone, and usually intersect with numerous other pathways

In enzyme pathways of several steps there are often catalyzed by key enzymes that are rate-limiting in controlling the flux through the pathway

This integrating expression of the pathway with other metabolic needs of the cell

Regulating Activity: 4 main strategies1. Allosteric control. Proteins contain distinct regulatory sites and multiple functional sites. Binding of regulatory molecules triggers conformational changes that affect the active sites. Display cooperativity: small [S] changes - major activity changes. Information transducers: signal changes activity or information shared by sites

Allosteric enzymes undergo conformational changes in response to modulator binding

Subunit interactions in anallosteric enzyme, and interactions with inhibitors and activators

Regulatory Enzymes

Hemoglobin allosteric properties

changes in quaternary structure lead to exposure of binding pocket thus easier access of O2

tertiary structure of low affinity: deoxy-Hb T (taut) state

tertiary structure of high affinity: HbO2 R (relax) state

Allosteric enzymes DO NOT obey Michaelis Menten kinetics!

At low substrate concentration, the enzyme is inactive thus in the in the inactive conformation.

As the substrate concentration is increased, substrate binds to enzyme and triggers a conformation change to the active conformation of the enzyme.

After the first substrate is bound, the second and subsequent substrates all bind more readily “cooperativity”

allosteric kinetics

Allosteric enzymes “S-shaped curve” indicates cooperative binding of substrate. The result of the change to the active shape is a steep increase in both substrate binding, and as a result, in reaction rate over a narrow range of substrate concentration.

When all active sites on the allosteric enzyme are occupied with substrate, a plateau is reached.

At low substrate concentration: the enzyme is in inactive conformation

As the substrate concentration is increased: substrate binds to enzyme and subsequent substrates all bind more readily

allosteric kinetics

Allosteric enzymes can be regulated between very low and very high reaction rates with only small changes in substrate concentration.

Allosteric enzymes are used by cells to regulate metabolic pathways where the concentration of cellular substrates fluctuate over narrow concentration ranges.

allosteric kinetics

aspartate transcarbamoylase ATC-ase

Two views of the regulatory enzyme ATCaseT R

This allosteric regulatory enzyme has two stacked catalytic clusters, each with three catalytic polypeptide chains (in shades of blue and purple) and three regulatory clusters, each with two regulatory peptide chains (in red and yellow).

Modulator binding produces changes in enzyme conformation and activity

Structure of ATCase - side viewSide view of quaternary structure

R and T states in equilibrium

ATCase does not obey MM kineticsSubstrate binding to one active site converts enzyme to R stateincreasing their activity: active sites show cooperativity

Basis of sigmoidal curveR & T states equivalent to 2 enzymes with different K0.5s

On the following plot, N represents the curve for an allosteric enzyme with no allosteric activators or inhibitors added. If an allosteric activator was added, which curve would one obtain?