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.
- How do G-proteins function?
G proteins are modulator proteins in cells that possess an internal clock. The clock is their GTPase activity that slowly hydrolyzes their own bound GTP to GDP and phosphate. As they hydrolyze GTP, their conformation changes and the complex they have formed with the target protein disassembles.
There are many G-proteins in any cell and the amount of time they stay active varies dramatically. Once activated by binding to GTP, the length of time any G-protein stays active depends upon its intrinsic GTPase activity. The GTPase activity of any G-protein has evolved to optimize its function.
For example, the role of the G-protein in the cAMP cascade:
- A hormone like adrenaline binds to a receptor on the plasma membrane and the conformation of the hormone receptor changes
- The new conformation allows binding of the G-protein and exchange of GTP for GDP. This binding of GTP activates G-protein.
- Acting as a modulator protein, G-protein binds to and activates its target protein, adenylate cyclase.
- ATP is converted to cyclic AMP by adenylate cyclase. Adenylate cyclase will continue to generate the second messenger cAMP from ATP as long as the activated G protein is bound.
- After a period of time determined by the intrinsic GTPase activity of the G-protein, the GTP is hydrolyzed and the reaction stops.
- What are zymogens?
Precursor proteins of proteases
- What are proteases?
Enzymes that cleave peptide bonds
- How are zymogens activated?
By proteolytic cleavage, which is the process of cleaving the zymogen into pieces by hydrolysis of peptide bonds
- Are chymotrypsinogen and prothrombin zymogens?
Yes.
Chymotrypsinogen is converted to chymotrypsin.
Prothrombin is converted to thrombin.
- What is the time frame of induction or repression?
Regulation by means of induction/repression of enzyme synthesis is usually slow in the human, occurring over 40-50 minutes, but usually hours to days. (Slow is in comparison to activation of a metabolic pathway that happens in seconds.)
- What is induction/repression?
Induction – signals genes to make more RNA to synthesize enzymes
Repression – signals genes to make less RNA and reduce enzyme synthesis
Induction and repression can refer to the synthesis of RNA and/or the synthesis of enzymes. Usually, when discussing metabolism, one is referring to enzyme induction or repression.
- Name one hormone that induces ubiquitin in muscle tissue. What is the result of ubiquitin induction?
Cortisol is the steroid hormone that is responsible for inducing ubiquitin (protein that binds to muscle protein) in muscle tissue. Cortisol is released in response to body stress, infection, fasting, or other systemic stress
Ubiquitin begins the muscle protein degradation process by tagging them for proteosomes. Proteosomes, in turn, catabolize proteins into amino acids that are used for gluconeogenesis, energy, or synthesizing new proteins
During fasting or infective stress, the concentration of cortisol in the blood and tissues rise. Cortisol causes induction of mRNA and an increase in ubiquitin synthesis. Ubiquitin is covalently attached to proteins destined to be catabolized into amino acids. The amino acids are then available for further use int eh body for antibody production, energy production from amino acids, and for use in gluconeogenesis by the liver
- Why is it important that the regulatory (control) enzyme for a pathway catalyze the rate limiting step in a pathway?
The control enzyme has the ability to increase or decrease the rate of the pathway by changing its enzyme activity. If it were not the slowest reaction, this would be impossible. The control enzyme is usually the first enzyme in a pathway.
- How is the rate of this step (enzyme) controlled?
The rate of a control enzyme may be changed by:
Availability of substrate
Product inhibition
Conformational changes
Allosteric
Covalent-modification
Proteolytic cleavage
Feedback inhibition (feedback inhibitors = allosteric inhibitors)
Enzyme induction (making more enzyme from more mRNA by increasing transcription
Enzyme repression (making less enzyme from less mRNA by decreasing transcription
- What does the term committed step mean?
The committed step in a pathway is a step (enzyme reaction) that is not reversible. Once the substrate is converted to product, it is doomed to finish the pathway. The committed step is catalyzed by the control enzyme.
- What are the two principal mechanisms for catabolizing ethanol in humans? Be able to write the reaction for the most common mechanism that uses NAD+ as a cofactor.
- MEOS: microsomal ethanol oxidizing system (part of the superfamily of cytochrome P450 enzymes). The activity of this pathway is minor in most individuals, but increases as a result of frequent, heavy alcohol use (such as in the case of Al Martini). It is an additional route for ethanol oxidation to acetaldehyde in the liver.
- Cytosolic alcohol dehydrogenase (most common mechanism):
Ethanol + NAD+ → Acetaldehyde + NADH + H+
AKA
CH3CH2OH + NAD+ → CH3CHO + NADH + H+
- Explain how increasing the amount of alcohol oxidized by alcohol dehydrogenase will affect the rate at which alcohol is oxidized. How does this affect fatty acid oxidation?
As ethanol is oxidized by the enzyme alcohol dehydrogenase, NAD+ is reduced to NADH, thereby increasing the concentration of NADH.
NADH is a product inhibitor of alcohol dehydrogenase, so when large amounts of NADH are produced, the rate of ethanol oxidation and ethanol clearance from the blood is decreased. That is, the more you drink, the slower you clear the alcohol.
High concentrations of NADH is also a product inhibitor of an enzyme in the pathway of fatty acid oxidation in the liver. Fatty acids come to the liver from adipose tissue to be oxidized, but as there is already such a high concentration of NADH, this oxidation/break down of fatty acids does not occur. Without this process, fatty acid content increases in the liver, and some speculate this might contribute to fatty liver disease in alcoholics.
- In which tissues do you find hexokinase and glucokinase and what is the reaction these enzymes catalyze?
Hexokinase is found in muscle
Glucokinase is found in the liver and beta cells of pancreas.
They both catalyze the same reaction (bc they are isozymes): Glucose + ATP → Glucose 6-P + ADP
- When glucose-6-phosphate inhibits hexokinase, is this product inhibition?
Yes, this is product inhibition. The product of the reaction inhibits the enzyme that created it. This happens by competition at the active site.
- Concerning Ann O’Rexia, what pathway is used when her cells wants to make energy from glucose-6-phosphate and how does the concentration of ATP or AMP in the cell affect the rate of this pathway? Is ATP a feedback inhibitor? Is ATP a negative allosteric effector and/or allosteric inhibitor? Is AMP a positive allosteric effector and/or allosteric activator?
Glycolysis is the pathway used to make energy from glucose-6-phosphate and the control enzyme for the pathyway is Phosphofructokinase-1.
High [AMP] increases the rate of glycolysis and high [ATP] decreases the rate of glycolysis
The ATP made by the pathway is a feedback inhibitor of the control enzyme. ATP is a negative allosteric effector and an allosteric inhibitor. The two terms are synonymous.
The AMP is a positive allosteric effector and an allosteric activator. The two terms are synonymous.
The concentration of ATP is high when the cell has enough energy
The concentration of AMP and ADP are high when the cell needs more energy
The total concentration of ATP, ADP, and AMP in the cell is constant so if ATP is low, AMP and ADP must be high, and vice versa.
- Concerning Ann O’Rexia, what pathway does she use for the storage of glucose as glycogen? How does glucose concentration and insulin affect this pathway?
Glycogen synthesis (aka glycogenesis) is the pathway she uses for the storage of glucose as glycogen.
High glucose and insulin concentrations activate this pathway in the liver and in the muscle cells.
- Concerning Ann O’Rexia who suffers from anorexia, if jogging activates both glycogenolysis and glycolysis, why does Ann tire easily?
Inadequate daily caloric intake prevents glycogen synthesis (glycogenesis), causing glycogen stores to run too low to support high energy activities such as jogging/running. When Ann starts running so does glycogenolysis, but the increased energy requirements soon outstrip the available glycogen.
- Concerning Ann O’Rexia, when she begins to jog, what muscle enzyme in glycogenolysis is activated by AMP? What type of activation is this? How does epinephrine activate this same enzyme? What role does cAMP and protein kinase A play in this cascade?
Glycogen phosphorylase is the muscle enzyme in glycogenolysis that is activated by AMP. This is positive allosteric activation.
Epinephrine (adrenaline) activates the cAMP cascade that eventually phosphorylates (activates) Glycogen phosphorylase.
- Concerning Ann O’Rexia, when she begins to jog, what enzyme in glycolysis is activated? What type of activation is this?
Phosphofructokinase-1 is the control enzyme in glycolysis that is activated by the increase in the AMP concentration. This is allosteric activations.
Although glycolysis is activated by positive allosteric activation, it will not run adequately due to lack of substrate (glucose-6-phosphate). Glycogenolysis would provide glucose-6-phosphate in a normal person but she has a lack of glycogen stores. As per an earlier objective, she will tire out quickly.
