Week 4 Flashcards
What are the three types of reversible inhibition?
Competitive, uncompetitive, noncompetitive
What is competitive inhibition?
Inhibitor competes with the substrate for the active site
What is uncompetitive inhibition?
Inhibitor binds to the enzyme-substrate, preventing product
What is noncompetitive inhibition?
The inhibitor binds to the allosteric site, changing the active site so the substrate isn’t able to bind
What does it mean for inhibition to be reversible?
The inhibitor binds then releases
What happens to the Km and Vmax during competitive inhibition?
Km increases, Vmax doesn’t change
What happens to the Km and Vmax during uncompetitive inhibition?
Km lowers, Vmax lowers
What happens to the Km and Vmax during noncompetitive inhibition?
Km doesn’t change, Vmax lowers
What is the effect of increased Km on binding?
Binding isn’t as good
In what situation is product formed during uncompetitive inhibition?
When the enzyme and substrate are bound, before the inhibitor binds
During non-competitive inhibition, what happens to the Km when the inhibitor molecule binds to the enzyme? When the substrate binds after?
Km increases, substrate binding decreases. Km decreases, substrate binding enhances
What happens when Vmax increases?
Rate of product formation increases
What does the lineweaker-burk plot look like for competitive inhibition?
What does the lineweaker-burk plot look like for uncompetitive inhibition?
What does the lineweaker-burk plot look like for noncompetitive inhibition?
In competitive inhibition, why doesn’t the Vmax change?
It can be overcome by a sufficiently high concentration of substrate
Identify competitive, noncompetitive, and uncompetitive inhibition from the Michaelis-Menten plot
What are the key features of irreversible inhibitors?
- Bind very tightly to active site
- No product forms, shuts the enzyme down
- Elucidates the mechanisms of enzyme action by covalently bonding to the enzyme
- Group-specific reagents react with R groups of specific amino acids
What are the 4 catalytic strategies used by enzymes?
- Covalent catalysis - Active site contains nucleophile that covalently binds
- Acid-base - Molecule other than H2O donates or accepts a proton
- Metal Ion - Funciton in several ways, serves as an electrophilic catalyst
- Approximation and orientation - Brings two substrates together in an orientation that facilitates catalysis
Enzyme activity can be modulated by?
Temperature, pH, and inhibitory molecules
Explain the chymotrpsin mechanism
- Lower barrier H-bond stabilizes His so that it can deprotonate Ser.
- Once H+ is removed, Ser can act as a stronger nucleophile to attack electrophilic amide C.
- Tetrahedral oxyanion is stabilized by H-bonding interactions with the oxyanion hole
- Collapse of the tetrahedral intermediate and H+ transfer from His leads to the cleavage of the C-N bond. The N-terminal peptide is bound through acyl linkage to Ser
- A water molecule binds to the active site and attacks the acyl ester carbonyl
- The resulting 2nd oxyanion intermediate tetrahedral is stabilized via enthalpic interations with the oxyanion hole
- The N-terminal fragment is released and returns to its initial state
What is the purpose of the oxyanion hole?
Stabilizes the negative charge of oxygen atom by making H-bond w/ amine side chaines of ser & gly
What are the key features of myoglobin?
- 8 subunits
- Globular tertiary structure
- Hydrophobic part of heme buried in the center of the protein
- Hydrophilic part of heme exposed to the surface
What are the key features of the catalytic triad?
- Asp 102 stabilizes His
- His 57 pulls H from Ser
- Ser 195 acts as a nucleophile
What is Heme?
Prosthetic group which is a precursor to hemoglobin, which is necessary to bind oxygen in the bloodstream
Identify the key components of the heme
In a heme, how is the Fe held?
6 coordinate covalent bonds from the four nitrogens in the tetrapyrrole group
What are the key features of the 4th, 5th, and 6th coordination sites of Fe of a heme?
- 4th is occupied by nitrogens in the tetrapyrrole ring
- 5th is nitrogen from proximal histidine
- 6th is occupied by O2
Identify the distal, O2, and proximal section of the histidine
What is the key feature of the Nitrogen of the distal histidine?
It’s too far away to participate in coordinate covalent bonding
What is the function of the hydrogen attached to the N of the distal histidine?
Stabilizes O2 when it’s present and bound to Fe2+
When O2 isn’t present, what does the distal histidine do?
Protects Fe2+ from oxidation
What is oxidation?
Loss of an electron
What happens when Fe2+oxidizes in the distal histidine?
Protein denatures, cannot reversibly bind O2
What is the dissociation constant?
Kd, quantitative measure of the affinity of a ligand for its protein
What is the chemical reaction between myoglobin and oxygen?
Mb + O2 -> MbO2
What is the formula for the dissociation constant for myoglobin?
Kd = [Mb][O2]/[MbO2]
What is fractional saturation?
Y, proportion of myoglobin molecules that have bound to O2
What is the equation of fractional saturation for myoglobin?
What is Kd when Y=1/2?
Kd = O2
What happens to the ligand when Kd decreases? Why?
Greater affinity for its protein. As Km decreases, the greater the affinity of substrate for enzyme
When comparing hemoglobin to myoglobin, which has a higher affinity for oxygen?
Myoglobin
What is a key feature of hemoglobin?
It’s allosteric, has multiple subunits (quaternary structure)
What is the equation of fractional saturation (Y) for hemoglobin?
What is the relationship between the hill coefficient and cooperativity?
Bigger Hill coefficient, more cooperativity
What does cooperativity mean in regards to hemoglobin?
If one heme group in hemoglobin has a bound O2, it converts hemoglobin to a high-O2 affinity state, increasing the ability of the other heme groups to bind O2
What is a key feature of hemoglobins AA sequences?
They’re similar but not identical
Hemoglobin has how many subunits, alpha, and beta chains?
- 4 subunits (tetramer)
- 2 alpha chains, 2 beta chains
What are the differences between myoglobin and hemoglobin?
- 18% similar in primary structure
- Very similar in secondary and tertiary structure with 8 alpha helices
- Hemoglobin can undergo quaternary structure
- Common evolutionary origin
What’s the difference in structure of hemoglobin going from deoxyhemoglobin to oxyhemoglobin?
Iron gets smaller because it now shares electron density with oxygen and it’s pulled further up into the plane of ring making the binding energy more favorable
What happens during the formation of oxyhemoglobin in addition to Fe pulling up into the plane?
O2 pulls up the proximal histidine which occupies the 5th coordination site causing a conformational change in the quaternary structure
What’s another way to describe the transformaiton of deoxyhemoglobin to oxyhemoglobin?
T-state to R-state
Explain the respiratory system in regards to deoxyhemoglobin and oxyhemoglobin
- O2 comes in through the lungs into the arteries
- Hemoglobin transports O2 to the tissues where it’s used for aerobic metabolism in the mitochondria
- Dissolved O2 diffuses freely or is bound to myoglobin, aiding in the transport of O2 to the mitochondria
- CO2 is carried back to the lungs by hemoglobin or in plasma and is then released
How does the binding of 2,3-BPG affect the T state?
Stabilizes it, lowers binding affinity for O2 allowing hemoglobin to offload O2 into cells
What are the key features of 2,3 biphosphoglycerate?
- Small
- Highly charged
- Size of hole in deoxyhemoglobin is the perfect size for it and is lined with positively charged residues
What is the Bohr effect?
Stimulation of oxygen release by carbon dioxide and H+
What are the key features of the Bohr effect?
- CO2 easily moves across membranes (small, nonpolar)
- CO2 becomes an acid in H2O which then undergoes acid-base equilibrium to produce H+
What is the relationship between pH, H+, and O2
-, +, more O2 will be released
Lowered pH allows the formation of what in regards to hemoglobin?
Formation of ionic interactions stabilizes the T-state, enhancing O2 release
What is the relationship between CO2 and hemoglobin?
CO2 reacts with terminal amino groups to form negatively charged carbanate groups which form salt bridges, stabilizing the T-state
What are CO2 and H+ to O2?
Heterotropic regulators, binding by Hb
Describe the relationship of pCO2, H+, pO2, and O2 in both tissues and lung
In tissues: +,+,-, oxygen is released
In lungs: -,-,+, oxygen is acquired
In what form is CO2 transported from tissue to lungs?
Bicarbonate
How does CO2 get transformed into bicarbonate?
Carbonic anhydrase
What is the function of allosteric effectors?
Bind to their target proteins and promote a conformational change that changes the functional properties of the protein
What is the diffference between homotropic allosteric and heterotropic allosteric effectors?
Homotropic ae’s bind at the active site whereas heterotropic ae’s bind at other sites on the protein
What are the positive and negative effectors for hemoglobin?
- O2 is a positive homotropic effector, increasing the binding affinity of O2 to hemoglobin
- H+, CO2, 2-3 BPG are negative heterotropic effectors, decreasing binding affinity
What is HbA, HbS, and HbF?
HbA is normal adult Hb
HbS - sickle cell
HbF - fetal Hb
What causes sickle cell?
Point mutation, AA, on beta chain
Where does HbF lie on the fractional saturation vs. pO2 graph? What does that mean?
In between myoglobin and hemoglobin. HbF has a higher affinity for O2 than Hb, but lower than myoglobin
What occurs during carbon monoxide poisioning?
CO is a competitive inhibitor, leading to increased O2 affinity on the other subunits, so Hb will not release enough O2
