Allosteric enzymes and Inhibitors

What is the Michaelis Menten model?

• a model of enzyme kinetics that shows the relationship between the substrate concentration and the rate of an enzyme-catalysed reaction

• Rate of reaction = velocity

• At the initial velocity (Vo) = no product (P)

• Product formation slows as we reach equilibrium. (Vmax)

Equations and

And…..

Vo = Vmax x S/Km+S

Km = K-1 + K2/K1

• at low S, a doubling of S will double Vo

• At a high S, increases in S will have little effect on Vo (enzyme is saturated)

What do the K’s mean?

Km = substrate concentration at ½ Vmax (high Km = low affinity for S, low Km = high affinity for S)

K1 = association rate constant

K2 =dissociation rate constant

K3 = catalytic rate constant

Assumptions of MMM

• binding is fast(ES)

• Catalysis is slower and rate-limiting

• At time 0, (Vo), P = 0

• Enzymes exist as either free or substrate bound

• Total enzyme(Et) = E+ES

• Rate of formation and breakdown of ES is equal during the steady state

What is a lineweaver-Burke plot?

• double-reciprocal graph

• X= reciprocal (-1/Km) of substrate conc

• Y= reciprocal (1/Vmax) of velocity

Reversible competitive inhibitors

• binds reversibly to the active site

• Non-covalent binding, dissociates from E

• High [S] outcompetes inhibitor from active site

Effect on Vmax = no change

Effect on Km = increases

Reversible non-competitive inhibitors

Type of mixed inhibitor.

• binds reversibly to site other than active site

• Changes enzymes structural conformation and reduces catalytic activity

• Affinity of enzyme for substrate unchanged

• Non-covalent binding

Effect on Vmax = decreases

Effect on Km = no chnage

Mixed inhibitors

• binds reversibly to allosteric site

• Binds to E and ES complexes

If inhibitor has higher affinity for E, acts like competitive inhibitor, and decreases Km

If inhibitor has higher affinity for ES, will act uncompetitive, and Km will increase

Vmax decreases

Reversible uncompetitive inhibitors

• binds to the ES complex and doesn’t allow product formation

• Slope (Km/Vmax) not affected

• Non-covalent binding

Effect on Vmax = decreases

Effect on Km = decreases

Irreversible inhibitors

• permanently deactivate denatured enzyme

• Covalent bond to active site

What do the reversible inhibitors bind to?

Competitive = only binds to free enzymes

Non-competitive = bind to free enzymes and ES complexes

Un-competitive = only binds to ES complexes

Lineweaver Burke plot - Competitive

No Vmax change, increased Km (affinity for S)

• increase in Km means lower affinity for S

Lineweaver Burke plot - non-competitive (Mixed)

No change in Km, pivots around Km, decrease in Vmax

Lineweaver Burke plot - uncompetitive

Decreased Vmax and Km

Lineweaver Burke plot - mixed

Decresed Vmax, either increase or decrease Km

What regulates enzyme activity?

• allosteric control

• Proteolytic activation of zymogens (inactive to active)

• Transcriptional regulation → amount of E expressed and rare of mRNA degradation

• Reversible covalent modification → kinases/phosphatases

• Tissue specific expression of isoenzymes (different but similar AA sequences but catalyse same reactions)

Allosteric enzymes

DONT follow Michaelis Mentem kinetics

• Allostery - catalytic activity altered by binding of effector/modulator to a site other than a the active site

• Homotropic allosteric effector = same as substrate (multiple subunits and active sites) e.g. O2 for Haemoglobin

• Heterotropic allosteric factor = regulatory molecule that is NOT the substrate, binds reversibly to active site e.g. CO2 for Haemoglobin

Homotropic effectors affect cooperativity:

• Cooperativity = +ve (increased binding on active site) or -ve (decreased binding on active site)

Haemoglobin as an example of effectors

O2→ binds to catalytic sites, changes the structure so more O2 binds more easily = Homotropic effector

CO2→ binds to non-catalytic sites, decrease Haemoglobin affinity for O2

Models of Allostery

MWC model

Concerted:

• subunits exist only in 2 states- T (compact) or R (relaxed)

• All subunits must be in the same state

• Equilibrium shifts to the R-state as more S binds

Sequential:

• substrate binding increases affinity without conformational chnage of whole enzyme

Sigmodial curve

Allosteric enzymes show cooperativity, so their activity can chnage based on substrate binding

Produces a different curve than MM kinetics

In metabolic pathways, the end product often allosterically inhibits the first enzyme (feedback intuition)

Regulation by covalent modification

Post-transcriptional modification:

• phosphorylation by Ser/Thr Kinase

• Reversed by action of protein phosphotases

Other:

Adenylation = transfer of adenylate from ATP

Acetylation e.g. Histones