Lecture 6: enzyme regulation and inhibtion

Define enzyme inhibitors

Molecules interfering with enzyme activity, slowing/ halting catalysis

What is the hierarchy of inhibition

Specific → Reversible → Competitive, Non-competitive and Uncompetitive

Specific → Irreversible

Non-Specific

What is specific inhibition

Only one type of enzyme inhibited

Reversible reaction meaning

The EI binding complex is in equilibrium

And non-specific

Inhibits al enzymes, doesn’t discriminate

What is KI

The inhibitor constant - indicates how potent an inhibitor is (how much EI is formed)

Does a competitive inhibitor permanently inactive the enzyme

No

What is the action of a competitive inhibitor

Competes with substrate for the active site, only I or S can be bound at one time

Write out Michaelis-Menten

V0 = Vmax [S] / aKm + [S]

What does a mean (equation)

a = 1 + [I]/KI

What does aKm mean

The observed Km in the presence of an inhibitor (called the apparent Km)

What happens if [S] » [I]

The reaction will be unaffected and will tend towards original Vmax as it is more likely that the substrate will bind than the inhibitor

What happens to Km in the presence of a competitive inhibitor

Increases

What happens to Vmax

Unchanged

Why is ethanol used as a competitive inhibitor for alcohol dehydrogenase

It binds instead of methanol, so formaldehyde (very toxic) isn’t formed as they have the same functional group (CH2OH) so both fit into the active site

Where does an uncompetitive inhibitor bind

A site distinct from the substrate active site and binds only to the ES complex

Can the substrate in an ESI complex be converted to product

No

What does KI’ mean

Different inhibitor complex

What is the equation for KI’

[ES] [I] / [ESI]

What does the Michaelis Menten equation look like for Uncompetitive inhibition

V0 = Vmax [S] / Km + a’ [S]

What does an uncompetitive inhibitor do to Km

Lowers it

Why is this

The inhibitor is removing some fraction of enzymes from the reaction (only ES removed which shifts binding EQ to right)

What is the action of a non-competitive inhibitor

Binds to both the free enzyme and the enzyme-substrate complex at a different site to the substrate

The inhibitor alters the structure of the protein in a way that prevents catalysis but doesn’t affect substrate binding

Michaelis-Menten equation for non-competitive inhibitors

V0 = Vmax [S] / aKm + a’[S]

Can the substrate still bind to the EI complex

Yes, but the ESI complex doesn’t progress to product

What does the inhibitor lower the concentration of

The functional enzyme

This therefore decreases

Vmax

What happens to Km

It remains the same as the substrate affinity for the enzyme is unchanged

What is the lineweaver burk plot for competitive inhibitors

Non-competitive?

Uncompetitive?

What does irreversible enzyme inhibitors do

They form permanent covalent interactions with a functional group on the enzyme which is essential for activity

What are mechanism-based irreversible inhibitors

The inhibitor binds like a substrate, catalytic mechanism starts but the catalytic process stalls as the inhibitor-enzyme interactions are too strong

What does it mean for enzyme activity to be spatiotemporally controlled

Means they have to catalyse reactions in the correct space at the correct time

What are the 4 types of regulation

Allosteric regulation

Reversible Covalent Modification

Proteolytic Cleavage

Feedback Regulation

If a product is in excess, how can enzymes be regulated

They can divert resources elsewhere

If a product is in demand, how can enzymes be regulated

They are activated to produce more of the required biomolecule

How do allosteric regulators (modulator/effectors) work

They bond to another site away from the active site on the enzyme, which can either activate or deactivate it through a conformational change

What is the difference between non-competitive inhibitors and allosteric regulators

Non-competitive is always inhibitory, whereas allosteric modulation can both inhibit or activate (doesn’t follow Michaelis-Menten

What is reversible covalent modification (or post-translational modification)

The enzyme activity function is modulated by covalent modification of amino acid residues in an enzyme molecule

What is an example of reversible covalent modification

Kinases add phosphate groups, phosphatases remove them

Enzymes regulates by proteolytic cleave are produced as

Inactive, called zymogen or pro-enzymes

How does it get activated

After removal of a polypeptide segment by proteolytic cleavage which causes conformational changes that form a fully functional enzyme (i.e. revealing active site)

What is feedback regulation

The end-product of an enzymatic pathway inhibits an upstream enzyme to decrease rate of production