Chp 9: Regulation of Enzymes Flashcards
- What are the terms found in the Michaelis-Menten equation and what do they mean?
Vi – the initial velocity, the initial rate of the reaction at a certain substrate concentration (the first few seconds of reaction)
Vmax – the maximal velocity (rate) a reaction can achieve at an infinite concentration of substrate
KM – the substrate concentration at which the reaction rate is at half-maximum and is a measure of the substrate’s affinity for the enzyme. A small KM indicates high affinity, meaning that the rate will approach Vmax at lower concentrations of substrate. The S0.5 is used in place of KM when dealing with allosteric enzymes (sigmoidal S-shaped curves)
[S] – substrate concentration. The rate of the reaction is dependent on the amount of substrate
- What kind of curve is derived for the MIchaelis-Menten equation?
Rectangular hyperbola
- Are glucokinase and hexokinase isozymes?
Yes
- Is hexokinase a Michaelis-Menten enzyme?
Yes, because it yields a rectangular hyperbola
- Is glucokinase of liver or pancreas a Michaelis-Menten enzyme?
No, because it yields a sigmoidal curve
- How does the S0.5 for pancreatic glucokinase in some patients with MODY compare with normal patients?
The S0.5 for pancreatic glucokinase in some patients with MODY is higher than it is for normal patients secondary to a mutation of glucokinase
- What effect does a higher S0.5 have on patients with MODY?
Insulin production is less than it should be for any level of blood glucose. For any level of glucose, the enzyme with a higher S0.5 will phosphorylate less glucose to glucose-6-phosphate. The release of insulin from these cells is dependent upon the release of glucose-6-P formed → less insulin is released.
Less insulin means less uptake of glucose from the blood to the cells, so blood glucose rises.
- What is the effect of a competitive inhibitor on the KM and Vmax?
KM increases while Vmax stays the same. The [S] has to be higher to compete with the competitive inhibitor to saturate the enzyme.
Vmax (rate of reaction) is not affected as the necessary amount of substrate is provided.
- What is the effect of a noncompetitive inhibitor on the KM and Vmax?
KM remains the same and Vmax decreases.
So the amount of substrate doesn’t change but since there are fewer enzymes available (either the noncompetitive inhibitor is blocking an active site in a multi-substrate reaction (pg 139) or it has bound so strongly that no amount of substrate can remove it – in whichever case, there are less enzymes available and thus the reaction velocity (Vmax) will decrease.
- How does product inhibition of hexokinase in one cell benefit all the other cells of the body?
Product inhibition is a decrease in the rate of an enzyme caused by accumulation of its own product. The product inhibition of hexokinase in one cell benefits all the other cells in the body by leaving glucose in the blood available for use by other cells according to their need.
Cells convert glucose to glucose-6-P → its concentration rises and inhibits hexokinase → glucose concentration rises:
hexokinase Glucose → Glucose-6-P glucokinase
Then the glucose in the cell rises to the level of glucose outside the cell so no more glucose enters the cell.
- What are the various names for the compounds that bind to an allosteric site? What effect do they have on the enzyme?
Allosteric activators or positive allosteric effectors/modulators
Allosteric compounds that decrease activity are called allosteric inhibitors or negative allosteric effectors/modulators
Both types bind to the enzyme at an allosteric site and stabilize a conformation of the protein.
Allosteric activators increase substrate binding and reaction rate. This conformation is called the high-activity, high-affinity, relaxed or R-state.
Allosteric inhibitors decrease substrate binding and reaction rate. This conformation is called the low-activity, low-affinity, tense or T-state.
- The substrates of allosteric enzymes exhibit positive cooperativity. Explain positive cooperativity in terms of subunits, conformation, and activity of the active site.
Positive cooperativity occurs when binding of the first substrate molecule increases the affinity of the other sites for substrate.
Allosteric enzymes usually have two or more subunits, each with an active site.
Without bound substrate, the enzyme may be in either the T-state or the R-state. When in the R-state, substrates can bind. Once one molecule of substrate is bound, the other active sites have a higher affinity for substrate so they are more likely to bind. The more substrate bound to active sites, the greater the enzyme activity.
Positive cooperativity results in a sigmoidal curve when Vi is plotted against concentration.
- What is the difference between the T-conformation (state) and the R-conformation of an allosteric enzyme?
T-state – low activity and low affinity for substrate; stabilized by allosteric inhibitors (inhibitors bind more tightly)
R-state – high activity and high affinity for substrate; stabilized by allosteric activators (activators bind more tightly) and by substrate
Activators bind in R-state, inhibitors bind in T-state
- Understand the effect that allosteric activators and inhibitors have on the conformation of an allosteric enzyme and on the plot of velocity versus substrate concentration. What about the S0.5?
Allosteric activators stabilize the R-state, slide the sigmoidal curve to the left, and lower the S0.5.
Allosteric inhibitors stabilize the T-state, slide the sigmoidal curve to the right, and increase the S0.5.
- What is the general name for the enzyme that places phosphate groups onto other enzymes?
Protein kinase
- What groups on enzymes are typically phosphorylated?
Serine and tyrosine
- What are the effects of phosphorylation?
Two effects of phosphorylation:
- Change in conformation, and thus the activity of a protein
- Creates a binding site for proteins with a complementary SH (src homology) domain
- What is the general name for the enzymes that hydrolyze and thus remove phosphate groups from proteins? Which bond is usually broken?
Protein phosphatase
Phosphoester bonds are broken
- What are the effects of dephosphorylation?
Dephosphorylation changes the conformation of the protein back to the state it was in before phosphorylation
- Explain how either AMP or phosphorylase kinase activates muscle glycogen phosphorylase.
Muscle glycogen phosphorylase has two states in the cell:
- Phosphorylase a is the active conformation (R-state)
- Phosphorylase b is the inactive conformation (T-state)
To change the enzyme to active form (high enzyme activity), AMP (a positive allosteric effector) must bind glycogen phosphorylase b at an allosteric site, and/or glycogen phosphorylase kinase must phosphorylate a seryl residue on glycogen phosphorylase b
- What is the effect of protein phosphatase upon phosphorylase a?
Protein phosphorylase hydrolyzes the phosphate groups from phosphorylase a, changing it back to the inactive state (phosphorylase b) unless AMP is still bound
- What are the activators of phosphorylase kinase in a muscle cell?
Adrenaline in the blood and the signal for muscle contraction:
Adrenaline:
- Binds to receptor on the cell membrane and after several steps increases [cAMP]
- [cAMP] binds to the regulatory subunit of Protein kinase A and releases the active catalytic subunit.
- Protein kinase A phosphorylates and activates Glycogen phosphorylase kinase
Muscle action potential:
- The muscle action potential signals for muscle contraction by increasing intracellular [Ca2+]
- The same increase in Ca2+ that activates contraction also causes increased binding of Ca2+ to calmodulin to form a Ca2+ calmodulin complex
- Ca2+ calmodulin complex, acting as a protein modulator protein, activates Glycogen phosphorylase kinase
- Starting with an increase in the concentration of cAMP that resulted from adrenalin binding to a receptor in the cell membrane, explain how phosphorylase is activated. How does the cascade result in the amplification of the original signal?
Adrenaline → Phosphorylase a
- Adrenalin (epinephrine) binds to its receptor on the cell membrane and after several steps increases [cAMP]
- cAMP binds to the regulatory subunit of Protein kinase A and releases the active catalytic subunit of Protein kinase A
- Protein kinase A phosphorylates and activates Glycogen phosphorylase b, converting it to the active form: Glycogen phosphorylase a
- Glycogen phosphorylase a is able to remove glucose units from glycogen and release glucose-1-phosphate into the cytosol.
Amplification:
- Binding of adrenalin to the receptor results in the creation of several thousand cAMP molecules that activate at least 1000 protein kinase A enzymes.
- Each protein kinase A enzyme phosphorylates at least 1000 Glycogen phosphorylase kinase enzymes.
- Each Glycogen phosphorylase kinase enzyme phosphorylates at least 1000 Glycogen phosphorylase enzymes
- So, the bind of one molecule of adrenalin was amplified 1000 x 1000 x 1000 = 1 billion times
- This is why several enzymes are used during certain processes (cascades). Because with a small hormone change outside a cell, a very large change can occur in a cell. If there was only one enzyme used then there would still be an amplification but not nearly as great
- Explain how an increase in calcium in muscle cells simultaneously activates muscle contraction and glycogenolysis. Which system uses ATP and which helps to produce ATP?
Muscle contraction (this system uses ATP)
- Muscle action potential triggers Ca2+ release inside the cell
- Ca2+ binds to troponin-C and removes inhibition between actin and myosin. Muscle contract converting a lot of ATP into ADP
Simultaneously:
- Glycogenolysis (this system helps to produce ATP)
- Muscle action potential triggers Ca2+ release inside the cell
- Ca2+ binds to the calmodulin subunit of muscle glycogen phosphorylase kinase forming the calcium calmodulin complex*
- Calcium-calmodulin complex activates glycogen phosphorylase kinase
- Activated glycogen phosphorylase kinase phosphorylates glycogen phosphorylase
- Glycogen phosphorylase degrades glycogen into glucose-1-phosphate
- Glucose-1-phosphate is converted into glucose-6-phosphate that goes through the glycolytic pathway, which generates ATP to supply energy for muscle contraction
*In most cases, calcium binds to calmodulin before the calcium-calmodulin complex binds to and activates proteins. In muscle, the calmodulin is permanently bound to the glycogen phosphorylase kinase.
When we contract our arm muscle, calcium is released into the cytosol like crazy so the calcium concentration increases in the muscle cell. The calcium binds to Troponin C (a protein that sits between the muscle fibers). The muscle fibers don’t touch each other because the Troponin C is in the way. When calcium binds to Troponin C it moves out of the way and the muscle is able to contract since now the muscle fibers can slide over each other. This process takes a lot of ATP, so while the muscle is contracting, the calcium tells the muscle cell to make more ATP through glycogenolysis – a coordinating pathway.