Enzymes need control mechanisms to prevent them from being constantly active or inactive.
Analogous to a car needing brakes and an accelerator.
Enzymes respond to environmental factors by speeding up or slowing down.
Allosteric Enzymes
Have more than one binding site:
Active site: where the substrate binds.
Allosteric site: where a regulatory molecule binds.
Switch between two conformations: active and inactive.
Binding of a regulatory molecule (ligand) changes the activity by stabilizing one of the two conformations.
Aspartate Transcarbamoylase
Regulated by the binding of cytosine triphosphate (CTP).
CTP binding to regulatory sites switches the enzyme to an inactive form.
Illustrates allosteric regulation.
Kinetics of Allosteric Enzymes
Allosteric enzymes may not follow Michaelis-Menten kinetics.
They often exhibit sigmoidal curves instead of hyperbolic curves, unlike Michaelis-Menten enzymes.
Feedback Inhibition
Metabolic pathways often involve a series of enzymes.
The final product of the pathway often inhibits an enzyme at the start of the pathway.
This is called feedback inhibition and prevents wasteful overproduction.
Mechanism of Feedback Inhibition
Once enough of the final product (F) is produced, it inhibits enzyme e1.
e1 is typically an allosteric enzyme regulated by the binding of F.
The regulated enzyme (e1) catalyzes the committed step in the pathway.
Aspartate Transcarbamoylase and Feedback Inhibition
Regulation of aspartate transcarbamoylase is a type of feedback inhibition.
The enzyme catalyzes the first step in CTP synthesis.
The product, CTP, inhibits aspartate transcarbamoylase by binding to the inactive form.
Allosteric Regulation by ATP and CTP
CTP is an allosteric inhibitor of aspartate transcarbamoylase.
ATP is an allosteric activator of aspartate transcarbamoylase.
The rate of N-carbamoylaspartate formation varies with aspartate concentration, and is affected by the presence of ATP or CTP.
Allosteric Proteins and Cooperativity
Show cooperativity and sigmoidal kinetics with regard to their substrate.
Bind ligands (effectors) that are not their substrates.
Effectors bind at effector binding sites, separate from the ligand binding site.
Effector binding influences the affinity of the protein subunits for the ligand.
Positive effectors increase affinity.
Negative effectors decrease affinity.
Effect of Effectors on Sigmoidal Curve
Positive effectors:
Steeper slope of the v (velocity) against [S] (substrate concentration) graph.
The v against [S] graph shifts from right to left.
Higher v for the same [S] until saturation.
Smaller change in [S] for the same change in v.
Negative effectors: Opposite effects.
Clinical Example - Oxygen Carrying Proteins
Myoglobin and hemoglobin.
Contain protein globin and the haem grouping.
The Fe^{2+} in the center of the haem group binds to oxygen; iron must be in the ferrous (II) oxidation state for effective binding.
Myoglobin
Stores oxygen in the muscle.
High saturation with O2 at pO2 of interstitial fluid, so sequesters O_2 into cell.
Releases O2 as pO2 falls during muscular activity.
Hemoglobin
Transports oxygen from the lungs to the tissues.
Adult hemoglobin (HbA) made up of 4 subunits: 2 α subunits and 2 β subunits, each with a haem group.
A molecule of hemoglobin can bind four molecules of O_2.
Hemoglobin is an allosteric protein exhibiting cooperativity of binding for oxygen.
The binding curve is sigmoidal in shape.
Hemoglobin Oxygen Binding
High saturation with O2 at pO2 of lungs, but low saturation at pO_2 of tissues.
Releases O2 to tissues due to lower pO2.
Sigmoid curve demonstrates cooperativity.
2,3-Bisphosphoglycerate (BPG)
Pure HbA in a test tube binds O_2 more efficiently than HbA in the blood due to the presence of BPG in the blood.
BPG acts as a negative effector, resulting in more O_2 delivered to the tissues.
BPG concentrations in blood increase when insufficient oxygen is getting to the tissues (e.g., cardiopulmonary insufficiency, high altitude).
Hemoglobin and BPG
In the presence of the negative effector BPG, hemoglobin releases more O_2 to the tissues than in the absence of the effector.
Foetal Hemoglobin (HbF)
Made of 4 subunits: 2 α and 2 γ instead of 2 β.
Binds oxygen more strongly than HbA because it has a lower affinity for BPG.
In the placenta, both maternal and foetal tissue are at the same pO_2.
HbF must be able to bind O2 more tightly than HbA so that O2 is transferred from maternal blood to foetal blood.
HbF and Oxygen Transfer
The lower affinity for BPG by HbF reduces sigmoidicity of the curve and allows it to take up O2 from HbA at pO2 in the placenta.
HbF in blood + BPG has a higher % Saturation than HbA in blood + BPG at the pO_2 of tissues, allowing O2 transfer.
HbF Efficiency
HbF is very efficient for the uptake of O_2 across the placenta.
HbF is not as efficient as HbA in transferring O_2 from lungs to tissues.
A few weeks before birth, γ chains cease to be produced and are replaced by β chains.
Until all HbF in neonatal blood is replaced by HbA, the infant has lower efficiency than an adult at transferring O_2 from lungs to tissues.
To counteract this, neonates/infants have a higher total Hb concentration in blood.
Models of Allosteric Action
Two models describe allosteric effects:
Concerted model.
Sequential model.
Probably a mixture of both in most enzymes.
Concerted Model (Monod, Wyman, Changeux)
Subunits can exist in only two forms: T (low affinity) and R (high affinity).
Cannot have mixed molecules (i.e., subunits are either all T or all R).
Cooperativity in Concerted Model
When the substrate binds to the T form, it causes all subunits to convert to the R form.
Because the R form has a higher affinity for the substrate than the T form, enzyme activity increases rapidly after each enzyme molecule has bound one substrate molecule.
Sequential Model (Koshland)
Subunits can exist in two forms: T (low affinity) and R (high affinity).
Can have mixed molecules (i.e., some subunits can be T while others are R).
Cooperativity in Sequential Model
When the substrate binds to the T form, it causes the subunit to which it binds to convert to the R form and makes it easier for the substrate to bind to the other subunits.
Because the affinity for the substrate increases once one subunit has converted to the R form, activity increases rapidly after the protein has bound one substrate molecule.
Summary of the Models
In both models:
Positive effectors stabilize the high affinity form of the subunits.
Negative effectors stabilize the low affinity form of the subunits.
Enzyme Kinetics and Inhibitors Summary
Some enzymes are allosterically regulated by inhibitors or activators.
Enzyme inhibitors can be reversible or irreversible.
Competitive reversible inhibitors increase apparent KM, but do not affect V{max}.
Noncompetitive inhibitors decrease apparent V{max}, but do not affect KM.
Irreversible inhibitors usually bind covalently to enzymes.