Chapter 5: Allosteric Enzymes Flashcards
Allosteric enzymes
- Oligomeric proteins
- Catalyze reactions toward the beginning of or following a branch point in metabolic pathways
Sigmoidal curve
- Allosteric enzyme plot
- Does not obey M/M kinetics
- V vs [S]
Active site
- Binding site for substrate
Allosteric site
- Binding site for small biochemical molecules
- Non-covalent binding
Allosteric effectors
- Binding site causes a subtle change in enzyme structure/shape
- Influences the ability of enzyme to bind substrate
Cooperativity
- The influence that the binding of a ligand to one subunit has on the binding of another ligand to a second subunit of an oligomeric protein
Homotropic effect
- The subtle conformational change in the second subunit may be induced by an allosteric modulator or by the substrate itself
Binding of ligand
- Causes changes in the quaternary structure of the protein
- Small changes in tertiary structure as well
T-State
- Less active conformation of allosteric enzymes
R-State
- More active conformation of allosteric enzymes
Sigmoidal curve when V vs [S] is plotted
- Generated due to the cooperative nature of substrate binding to an allosteric enzyme
- Binding of first substrate molecule enhances further substrate binding in homotropic way
Activators
- Heterotropic allosteric effectors
- Increase catalytic activity
Inhibitors
- Effectors that reduce/prevent substrate binding to active site
Allosteric activators
- The purine ATP in the case of ATCase
Allosteric inhibitors
- The pyrimidine CTP in the case of ATCase
Aspartate transcarbamoylase (ATCase)
- Composed of two catalytic trimers and three regulatory dimers
ATCase
- Catalyzes the first step in the pathway of pyrimidine nucleotide biosynthesis
- Committed step in bacteria
Cytidine triphosphate (CTP)
- End product of pyrimidine nucleotide biosynthesis pathway
- Inhibits ATCase
- Feedback/end-product inhibition
Phosphofructokinase 1 (PFK-1)
- Tetrameric allosteric enzyme
- Phosphorylates fructose 6-phosphate to fructose 1, 6-bisphosphate (committed step in glycolytic pathway)
Fructose 6-phosphate
- Substrate that binds in a positively, cooperative manner
PFK-1 kinetics
- Sigmoidal kinetics
- Sensitive to the energy level of the cell
PFK-1 most active
- At low levels of ATP
PFK-1 most inhibited
- At high levels of ATP
Curve when PFK-1 inhibited
- Substrate-binding curve shifts to the right
- Becomes more sigmoidal
ATP effects on PFK-1
- Serves as both a substrate and an allosteric inhibitor of PFK-1
Fructose 2, 6-bisphosphate
- The most potent allosteric activator of PFK-1
- Favors the T –> R transition
- Enhances the enzyme’s affinity for its substrate (fructose 6-phosphate)
Glycogen phosphorylase
- Catalyzes a key regulatory and irreversible step in glycogenolysis
- Active a-form
- Less active b-form
- Found in liver and muscle tissue
Glucose
- Acts as an allosteric inhibitor of glycogen phosphorylase in the liver
- Reduces the breakdown of glycogen when blood sugar levels are high
AMP in muscle tissue
- Serves as a potent allosteric activator of the enzyme (glycogen phosphorylase) favoring glycogenolysis
Protein Kinase A
- Tetrameric allosteric enzyme
- Two separate catalytic subunits (38kD)
- Two regulatory subunits that are catalytically inactive (49kD)
Intracellular cAMP
- Stimulates protein kinase A when elevated
- Causes tetramer dissociation into a regulatory dimer and two catalytically active monomers
Hemoglobin
- Tetrameric allosteric protein
- Consists of two structurally similar alpha and beta globin subunits
- Held together by hydrogen bonds and electrostatic interactions
- Each subunit: 8 alpha helical domains and a heme prosthetic group
Hemoglobin function
- Present only in erythrocytes
- Serves as an oxygen delivery system
Hemoglobin affinity
- Low affinity for oxygen in actively respiring tissues (readily giving oxygen up)
- High affinity for oxygen in the lungs
Myoglobin
- Monomeric protein
- Facilitates oxygen storage in actively respiring muscle
Oxygen-dissociation curve for myoglobin
- Hyperbolic curve
- Expected kinetics of a monomeric protein with a single heme oxygen-binding site that does not exhibit cooperativity
Molecular oxygen
- Binds reversibly to the heme group causing conformational changes
- Changes hemoglobin from T to R state
T-State of oxygen
- Deoxygenated state
R-State of oxygen
- Oxygenated state
Each hemoglobin subunit
- Binds one molecule of oxygen to the ferrous iron of the heme prosthetic group
Positive cooperativity of hemoglobin
- The binding of a single oxygen molecule to a subunit of deoxyhemoglobin increases the likelihood that subsequent oxygen molecules will bind to the adjacent hemoglobin subunits
Oxygen-dissociation curve for hemoglobin
- Plots fractional saturation (Yo2) on the y-axis
- Plots the partial pressure of oxygen on the x-axis (pO2)
Increasing pH on hemoglobin plot
- Shifts sigmoidal curve to the left
Lowering pH on hemoglobin plot
- Hemoglobin gives off oxygen more readily
- Shifts sigmoidal oxygen-dissociation curve to the right
Purified (stripped) hemoglobin
- Has a high affinity for oxygen
Negative allosteric effectors of purified hemoglobin
- D-2
- 3-bisphosphoglycerate (BPG)
- Carbon dioxide
- Hydrogen ions
Effect of negative allosteric effectors of purified hemoglobin
- Reduce its affinity for oxygen
- Stabilize the T-form (deoxygenated)
- Shift the sigmoidal oxygen-dissociation curve for purified hemoglobin to the right
Allosteric enzymes
- Possess two physically distinct binding sites that are cooperative
Allosteric effectors
- May be either activators or inhibitors, structurally unrelated to the enzyme substrate
Sigmoidal Vo vs [S] plot
- Shape of allosteric enzyme curve
- Indicates that the enzyme does not obey M/M kinetics
Important allosteric enzymes
- Phosphofructokinase-1 (of glycolysis)
- Glycogen phosphorylase
- cAMP-dependent protein kinase (protein kinase A)
Hemoglobin
- Not an enzyme
- Exhibits characteristics of allosteric enzymes
