biochemistry practice questions
Negative feedback often involves the final product and an enzyme that catalyzes an early reaction in the pathway. Briefly explain why this promotes efficiency.
By targeting the first step of the pathway minimize wasted intermediates.
Consider two enzymes at the same concentration. The enzymes have the same Vmax, but different Km values. For each statement below, circle true or false. True or False: The enzyme with the higher Km has a higher catalytic efficiency
False
True or False: Enzymes are consumed during each catalyzed reaction and can only act once.
False
True or False: Enzymes change the free energy of the reaction
False
True or False (circle one): The overall reaction catalyzed by hexokinase consumes energy in the form of ATP and the overall reaction catalyzed by glucose 6- phosphatase generates energy in the form of ATP.
False -Pay attention to the overall - meaning that it is talking about glycolysis and gluconeogenesis. Overall, glucose generates ATP and gluconeogenesis consumes ATP
Which curve represents fructose 1,6 bisphosphatase activity when [ATP] is high?
High activity means that the KM would be lower, which would be curve 1
*Mutations that disrupt the function of the glucose 6-phosphate transporter cause a human disease characterized by low blood glucose levels and increased amounts of glycogen in the liver. For each of these symptoms, explain how the non-functional glucose 6-phosphate transporter leads to these symptoms.* Low blood glucose levels: Increased amount of glycogen:
*Low blood glucose levels:* Glucose can't be made from glucose 6-phosphate (glycogen breakdown or gluconeogenesis) and released to blood when glucose levels are low *Increased amount of glycogen:* Extra glucose 6-phosphate in the cell made by gluconeogenesis will be converted to glycogen
Describe in your own words (not an equation) what the KM of an enzyme tells you about the enzyme activity.
-the [S] when reaction velocity reaches ½ Vmax -the [S] when E is 50% bound by S -the affinity of E for S -the stability of ES
Some snake venom phospholipases are catalytically inactive due to a mutation of Asp to Lys in the active site. What important role does Asp have in the active site of phospholipase and how does that contribute to the function of Phospholipase A2?
1- position histidine 2- make histidine more likely to abstract proton from water
Name 3 glycolytic enzymes catalyzing irreversible glycolysis steps
1. HEXOKINASE 2. PHOSPHOFRUCTOKINASE 3. PYRUVATE KINASE
Mutations that disrupt the function of the glucose 6-phosphate transporter cause a human disease characterized by low blood glucose levels and increased amounts of glycogen in the liver. For each of these symptoms, explain how the non-functional glucose 6-phosphate transporter leads to these symptoms. 1. Low blood glucose levels 2. Increased amount of glycogen
1. Low blood glucose levels = Glucose can't be made from glucose 6-phosphate (glycogen breakdown or gluconeogenesis) and released to blood when glucose levels are low 2. Increased amount of glycogen = Extra glucose 6-phosphate in the cell made by gluconeogenesis will be converted to glycogen
What is the value of alpha for molecule Y?
KM apparent = alpha x KM
PFK1 is considered an inefficient enzyme. How would you define catalytic efficiency (using words or kinetics expression)?
Kcat/Km. How often an interaction results in a conversion of substrate to product
Why is k-2 not included in the Michaelis Menten model?
Michaelis Menten kinetics is based on measurement of the initial velocity (V0) which is the velocity of the reaction at the very beginning of the reaction when there is very little product and therefore no reverse reaction (conversion of product into substrate).
Trypsin is a serine protease that cuts proteins after Lys or Arg amino acid residues. How would you predict that a mutation of Asp to Gly in the specificity pocket of trypsin would be most likely to change the activity of the enzyme? Select one answer. a. KM would increase b. KM would decrease c. Vmax would increase d. Vmax would decrease
Mutation of Asp to Gly would increase Km (decrease affinity of substrate) because specificity pocket is involved in binding to the substrate. More specifically, the Asp forms an ionic interaction with Lys and Arg. Gly is not charged and can not form an ionic interaction so the mutant enzyme will not bind as well to substrate.
The serine protease mechanism includes an acyl-enzyme intermediate in which the substrate is covalently attached to the enzyme. Do you expect the phospholipase mechanism to include this as well?
No. A water molecule is the nucleophile, so none of the residues of the enzyme directly bond to the substrate during the reaction. Thus, there is no acyl-enzyme intermediate.
In a cell in which glycolysis is continuously active, do you think the reaction 3- phosphoglycerate <------> 2-phosphoglycerate will reach equilibrium?
No. There will be not much of reverse reaction occurring because the next product is depleted (PEP) by the irreversible conversion to pyruvate. Irreversible reactions deplete intermediates so reactions do not proceed in the reverse direction and equilibrium is not reached.
In a cell in which glycolysis is continuously active, do you think the reaction 3- phosphoglycerate <------> 2-phosphoglycerate will reach equilibrium?
No. There will be not much of reverse reaction occurring because the next product is depleted (PEP) by the irreversible conversion to pyruvate. Irreversible reactions deplete intermediates, so reactions do not proceed in the reverse direction and equilibrium is not reached.
Different snakes contain different versions of PLA2 that cut the same types of bond using the same catalytic mechanism, but have different phospholipid substrates. Explain how two enzymes with the same active site can have different substrates.
Parts of the substrate that are not changed by the reaction are recognized by other parts of the enzyme (specificity pocket)
Describe what is happening to substrates and products of the reaction during the time indicated by the arrow on the graph. (Line levels out)
Reaction is approaching equilibrium when forward rate (production of product) equals reverse rate (production of substrate)
Using Michaelis-Menton kinetics to describe enzymes requires 1) the steady state assumption and 2) measurement of initial velocity (V0). Choose one of these and explain what this means and why this is important to consider when measuring Michaelis-Menton kinetics.
Steady-state assumption: [ES] is stable or has reached equilibrium or rate of formation is equal to breakdown; this simplifies the situation that needs to be described and allows the concentrations and rates to be used to derive Km Initial velocity: Rate is measured at the initial velocity so that product is not building up and the conversion of product to substrate (or k-2) does not need to be considered.
True or False: Enzymes bind to their substrates and products
True
True or False: Many enzymes change shape slightly when substrates bind
True
True or False: Reactions occur at the active state of enzymes where a precise 3D orientation of amino acids is an important feature of catalysis
True
Consider two enzymes at the same concentration. The enzymes have the same Vmax, but different Km values. For each statement below, circle true or false. True or False: The enzymes have the same kcat
True Vmax is equal to the product of the catalyst rate constant (kcat) and the concentration of the enzyme
*Pyruvate kinase catalyzes the conversion of phosphenolpyruvate (PEP) to pyruvate in glycolysis.* *What prediction would you make about the regulation of pyruvate kinase? Select all that apply.* a. Fructose 1,6-bisphosphate stimulates pyruvate kinase activity b. Glucose 6-phosphate inhibits pyruvate kinase activity c. Phosphoenolpyruvate inhibits pyruvate kinase activity d. Pyruvate stimulates pyruvate kinase activity
a. Fructose 1,6-bisphosphate stimulates pyruvate kinase activity
*Fructose 1,6-bisphosphatase and phosphofructokinase-1 are reciprocally regulated. Which statements are true about the reciprocal regulation? Select all that apply.* a. glycolysis and gluconeogenesis will not occur in a liver cell at the same time b. if glycolysis is occurring in muscle cells, gluconeogenesis cannot occur in liver cells at the same time c. if glycolysis is not occurring in a liver cell, then gluconeogenesis will be occurring in the liver cell
a. glycolysis and gluconeogenesis will not occur in a liver cell at the same time *(reciprocal regulation = the amts and activities of the enzymes involved in both pathways are controlled so that the pathways are not highly active at the same time; intended to prevent concurrent activity in two closely parallel pathways)*
Fructose 1,6-bisphosphatase (F16BPase) and phosphofructokinase-1 (PFK1) are both regulated by binding to fructose 2,6 bisphosphate (F26BP). Which statements are true? Select all that apply. a. if fructose 2,6 bisphosphate levels are high, glycolysis will be activated b. if fructose 2,6 bisphosphate levels are high, gluconeogenesis will be activated c. phosphofructokinase-2 (PFK2) activity leads to activation of phosphofructokinase-1 (PFK1) d. fructose 2,6 bisphosphate is a competitive inhibitor of phosphofructokinase-1 e. fructose 2,6 bisphosphate is an activator of fructose 1,6-bisphosphatase
a. if fructose 2,6 bisphosphate levels are high, glycolysis will be activated *(in the chart on class slides)* c. phosphofructokinase-2 (PFK2) activity leads to activation of phosphofructokinase-1 (PFK1)
*If protein kinase A (the same kinase that regulates pyruvate kinase) is activated, glycogen phosphorylase would be: Select one answer.* a. in the more active state so glycogen breakdown increases. b. in the less active state so glycogen breakdown increases. c. in the more active state so glycogen breakdown decreases. d. in the less active state so glycogen breakdown decreases
a. in the more active state so glycogen breakdown increases.
*Pyruvate kinase is a substrate of Protein kinase A. Which statement is TRUE if protein kinase A is active? Select one answer.* a. increases phosphorylation and inhibits activity of pyruvate kinase b. increases phosphorylation and stimulates pyruvate kinase activity c. decreases phosphorylation and inhibits pyruvate kinase activity d. decreases phosphorylation and stimulates pyruvate kinase activity
a. increases phosphorylation and inhibits activity of pyruvate kinase *(protein kinases catalyze phosphorylation, which means activity of pyruvate kinase is inhibited - phosphorylation is the process of introducing a phosphate group into a molecule - as seen in the diagram, the phosphate group on pyruvate kinase turns off its activity)*
*Which statement(s) is/are true. Select all that apply.* a. phosphorylation affects allosteric regulation of glycogen phosphorylase by the molecules shown on the diagram b. allosteric regulation by the molecules shown on the diagram affects phosphorylation of glycogen phosphorylase c. the phosphorylated form of glycogen phosphorylase is always active and the unphosphorylated form is always inactive (no because there is still an active and inactive state for both the phosphorylated form and unphosphorylated form) d. phosphorylation of glycogen phosphorylase favors the R-state of the enzyme e. [ATP] is the only factor that determines whether glycogen phosphorylase will be phosphorylated or unphosphorylated
a. phosphorylation affects allosteric regulation of glycogen phosphorylase by the molecules shown on the diagram d. phosphorylation of glycogen phosphorylase favors the R-state of the enzyme -*Phosphorylation of enzyme causes the R form to be stabilized-Normally it is present such that the T form is favored; non-phosphorylating - equilibrium favors the T state-Adding a phosphate shifts the equilibrium such that the R state is favored - adding one bond (phosphate bond) and shifting equilibria*
Name three different regulatory strategies used by enzymes that were described in lecture
allosteric control (inhibition), isozymes, covalent modifications (phosphorylation), proteolytic cleavage, control amount of enzyme by transcription or translation
Explain how affinity and reaction rate contribute to the catalytic efficiency. What must be true of an inefficient enzyme like PFK1?
an inefficient enzyme will either have a high Km (low affinity for substrate), a slow reaction rate (k2, kcat) or both.
*GLUT2 is a low affinity (high Km) glucose transporter found in pancreatic alpha and beta cells. What is the consequence of a mutation that changes GLUT2's Km for glucose from 5 mM to 1 mM? Select one answer.* a. Pancreatic alpha cells will release glucagon even if blood glucose level is high b. Pancreatic beta cells will release insulin even if blood glucose concentration is low c. Pancreatic alpha cells will release glucagon even if blood glucose level is low d. Pancreatic beta cells will release insulin even if blood glucose concentration is high
b. Pancreatic beta cells will release insulin even if blood glucose concentration is low *GLUT2 (Glucose Transporter 2) is a transmembrane protein that allows the movement of glucose in the cells of the liver, pancreas, kidneys and small intestine. This transporter has a low affinity but a high capacity for transporting glucose, meaning that glucose will only be transported across the membrane when it is in high concentration inside a cell - triggers release of insulin. Thus, in this case, even at low concentrations, glucose will be transported across the membrane.* Low affinity glucose transporter = release insulin High affinity glucose transporter = release glucagon
Vmax is a measure of the maximum velocity of a reaction. Under what conditions is Vmax reached? Select all that apply. a. When all S is bound to E b. When all E is bound to S c. When the rate of product formation equals the rate of the reverse reaction d. When there is no more S available to react e. When [S]
b. When all E is bound to S
*Some studies show that sucralose can increase insulin levels in the blood. Which enzymes would you expect to be activated by insulin and inhibited by glucagon? Select all that apply.* a. glycogen phosphorylase b. glycogen synthase c. phosphofructokinase-1 d. phosphofructokinase-2 e. fructose 1,6-bisphosphatase f. fructose 2,6-bisphosphatase
b. glycogen synthase *(forming glycogen from glucose (getting rid of glucose); if you were to choose (a), you would be producing glucose by breaking down glycogen)* c. phosphofructokinase-1 *(involved in glycolysis - breaking down glucose to make pyruvate)* d. phosphofructokinase-2 *(involved in glycolysis - breaking down glucose to make pyruvate)* *Insulin wants to remove glucose, so the enzymes that are involved in breaking down glucose / getting rid of glucose would be activated
*ATP is a substrate and a regulator of pyruvate kinase. Based on the overall glycolysis pathway, which of the following types of BEST explains how ATP regulates pyruvate kinase? Select one answer.* a. feedforward stimulation b. negative feedback inhibition c. competitive inhibition d. proteolytic cleavage e. irreversible inhibition
b. negative feedback inhibition *(one of the products downstream in a reaction series comes back and inhibits the enzymatic activity of an earlier reaction; a type of regulation in biological systems in which the end product of a process in turn reduces the stimulus of that same process)*
The Km of an enzyme for a substrate is: Select one answer. a. half the substrate concentration at which the reaction rate is maximal b. the substrate concentration at which the reaction rate is half maximal c. the rate constant of the reaction at half saturation d. the dissociation constant of the enzyme- product complex e. the rate constant of the reaction at saturation
b. the substrate concentration at which the reaction rate is half maximal
*CTP regulates the activity of aspartate transcarbamoylase (ATCase). ATCase catalyzes the second step of the pathway above. Which of the following are consistent with what you know about CTP regulation? Select all that apply.* a. CTP is likely to act as competitive inhibitor of ATCase b. CTP is an activator of ATCase c. CTP regulation of ATCase is an example of negative feedback inhibition d. CTP is likely to be an allosteric inhibitor of ATCase e. CTP binds to the catalytic subunit of ATCase (Class slides - CTP decreases catalytic activity of ATCase; stabilizes the T state of ATCase and increases the apparent KM of substrate binding; CTP and ATP allosterically regulate Aspartate Transcarbamoylase in opposite ways; Cytidine Triphosphate (CTP) is the end product of the reaction pathway)
c. CTP regulation of ATCase is an example of negative feedback inhibition d. CTP is likely to be an allosteric inhibitor of ATCase *(Class slides - CTP decreases catalytic activity of ATCase; stabilizes the T state of ATCase and increases the apparent KM of substrate binding; CTP and ATP allosterically regulate Aspartate Transcarbamoylase in opposite ways; Cytidine Triphosphate (CTP) is the end product of the reaction pathway)*
How do enzymes catalyze chemical reactions? Select all that apply. a. Decrease the standard free energy of reaction (G) b. Change the equilibrium constant (Keq) of the reaction c. Decrease the time it takes a reaction to reach equilibrium d. Decrease the activation energy of a reaction e. Increase the rate of the forward reaction (not the reverse reaction) f. Decrease the rate of the reverse reaction
c. Decrease the time it takes a reaction to reach equilibrium d. Decrease the activation energy of a reaction
*Which statement is true about Vmax? Select all that apply.* a. Vmax is the velocity when [S]<KM b. Vmax is the velocity of a reaction when [S]=[E] c. Vmax is the initial velocity of a reaction when [E]Total=[ES] d. Vmax is the maximum velocity of a reaction at a single enzyme concentration e. Vmax is the velocity at a single substrate concentration
c. Vmax is the initial velocity of a reaction when [E]Total=[ES] *(because initial velocity = Vmax when [E]Total = [ES])* d. Vmax is the maximum velocity of a reaction at a single enzyme concentration *(because you are not changing the enzyme, only the substrate concentration)*
Which statement is true about Vmax? Select all that apply. a. Vmax is the velocity when [S]<KM b. Vmax is the velocity of a reaction when [S]=[E] c. Vmax is the initial velocity of a reaction when [E]Total=[ES] d. Vmax is the maximum velocity of a reaction at a single enzyme concentration e. Vmax is the velocity at a single substrate concentration
c. Vmax is the initial velocity of a reaction when [E]Total=[ES] d. Vmax is the maximum velocity of a reaction at a single enzyme concentration
In the presence of a small molecule, the observed Vmax and the observed Km of the catalyzed reaction are decreased compared the reaction in the absence of the small molecule. How would you describe the effect of the small molecule on the enzyme? Select one answer. a. competitive inhibitor of the enzyme. b. mixed (noncompetitive) inhibitor of the enzyme c. uncompetitive inhibitor of the enzyme d. activator of the enzyme
c. uncompetitive inhibitor of the enzyme
What can you determine from this type of graph (no additional information except for values on the axes)? X axis = time Y axis = product a. Km b.Vmax c.V0 d. kcat e. [E] f. [S] g. none of the above
c.V0
*Consider a molecule of glucose 6-phosphate (G6P) in liver cell. What are the likely possible fate(s) for glucose 6-phosphate under conditions when blood glucose levels are high? Select all that apply.* a. G6P could be converted to glucose by hexokinase b. G6P could be converted to glucose by glucose 6-phosphatase (already high levels of glucose, so it will not be converted to glucose) c. G6P could exit the cell through glucose transporters (Gluts) d. G6P could be converted to fructose 6-phosphate by phosphohexose isomerase e. G6P could be converted to glucose 1-phosphate by phosphoglucomutase f. G6P could be converted to glycogen by glycogen phosphorylase
d. G6P could be converted to fructose 6-phosphate by phosphohexose isomerase *(already high levels of glucose, so makes sense that the pathway would go the opposite direction)* e. G6P could be converted to glucose 1-phosphate by phosphoglucomutase *(already high levels of glucose, so makes sense that the pathway would go in a direction that is not towards making glucose)*
*Under conditions when gluconeogenesis is active, how would you best describe the direction of net transport you would expect to observe for glucose-6-phosphate and Pi? Assume the glucose 6-phosphate transporter is the only membrane transporter present in the endoplasmic reticulum that can bind to glucose 6- phosphate and Pi. Select one answer.* a. Glucose 6-phosphate and Pi would move from the cytosol into the ER b. Glucose 6-phosphate and Pi would move from the ER into the cytosol c. Glucose 6-phosphate would move from the ER into cytosol and Pi would move from the cytosol into the ER d. Glucose 6-phosphate would move from the cytosol into the ER and Pi would move from the ER into the cytosol e. Glucose-6-phosphate and Pi would move independently across the membrane
d. Glucose 6-phosphate would move from the cytosol into the ER and Pi would move from the ER into the cytosol *1. Glucose-6-phosphatse is in the ER so G6P needs to move from the cytosol to the ER for the reaction to occur. 2. The reaction generates Pi which needs to move from the ER to the cytosol. 3. Because the type of transport is antiport, both molecules need to move together, but in opposite directions across the membrane.*
Under conditions when gluconeogenesis is active, how would you best describe the direction of net transport you would expect to observe for glucose-6-phosphate and Pi? Assume the glucose 6-phosphate transporter is the only membrane transporter present in the endoplasmic reticulum that can bind to glucose 6- phosphate and Pi. Select one answer. a. Glucose 6-phosphate and Pi would move from the cytosol into the ER b. Glucose 6-phosphate and Pi would move from the ER into the cytosol c. Glucose 6-phosphate would move from the ER into cytosol and Pi would move from the cytosol into the ER d. Glucose 6-phosphate would move from the cytosol into the ER and Pi would move from the ER into the cytosol e. Glucose-6-phosphate and Pi would move independently across the membrane
d. Glucose 6-phosphate would move from the cytosol into the ER and Pi would move from the ER into the cytosol 1. Glucose-6-phosphatse is in the ER so G6P needs to move from the cytosol to the ER for the reaction to occur. 2. The reaction generates Pi which needs to move from the ER to the cytosol. 3. Because the type of transport is antiport, both molecules need to move together, but in opposite directions across the membrane.
*In the presence of a small molecule, the observed Vmax and the observed Km of the catalyzed reaction are decreased compared the reaction in the absence of the small molecule.* *Which statement below is true? Select all that apply.* a. With the small molecule present, the enzyme would convert substrate to product faster. b. With the small molecule present, it would take more substrate to drive the reaction to half-maximum velocity. c. The small molecule can only bind to the enzyme when substrate is not bound d. The small molecule increases the apparent affinity of the enzyme for substrate e. The small molecule is an allosteric regulator of the enzyme
d. The small molecule increases the apparent affinity of the enzyme for substrate e. The small molecule is an allosteric regulator of the enzyme *(allosteric regulator = enzymes that change their conformational ensemble upon binding of an effector (allosteric modulator) which results in an apparent change in binding affinity at a different ligand binding site. This "action at a distance" through binding of one ligand affecting the binding of another at a distinctly different site, is the essence of the allosteric concept)*
Which statement below is true? Select all that apply. a. With the small molecule present, the enzyme would convert substrate to product faster. b. With the small molecule present, it would take more substrate to drive the reaction to half-maximum velocity. c. The small molecule can only bind to the enzyme when substrate is not bound d. The small molecule increases the apparent affinity of the enzyme for substrate e. The small molecule is an allosteric regulator of the enzyme
d. The small molecule increases the apparent affinity of the enzyme for substrate e. The small molecule is an allosteric regulator of the enzyme
Consider a newly-discovered serine protease that contains the catalytic triad D-H-S and cuts peptide bonds after hydrophobic residues. A. How would you expect mutation of the aspartic acid of the catalytic triad to glycine to affect the function of the enzyme? Select all that apply. a. decrease Km b. increase Km c. increase kcat d. decrease kcat e. none of the above
d. decrease kcat Glycine would not hydrogen bond with His leading to decreased formation of the serine nucleophile which would reduce the rate of reaction(kcat). Km would not be affected because Asp is not involved in binding the substrate. This change would affect kcat (catalytic rate), but not Km (binding of the substrate)
Trypsin is a serine protease that cuts proteins after Lys or Arg amino acid residues. How would you predict that a mutation that disrupts the function of the oxyanion hole of trypsin would be most likely to change the activity of the enzyme? Select one answer. a. KM would increase b. KM would decrease c. kcat would increase d. kcat would decrease
d. kcat would decrease The kcat would decrease because the oxyanion hole is involved in stabilizing the transition state which is directly related to the rate of catalysis. (The oxyanion hole is not involved in binding to the substrate so Km is not affected.)
When would you expect the activity of fructose 1,6 bisphosphatase to be high? Circle one: high [ATP] low [ATP]
high [ATP] *If we look at the end products of the overall pathway that 1,6 bisphosphatase is involved in (gluconeogenesis), we see that the pathway consumes energy (does not produce energy). Therefore, ATP would not inhibit the activity of fructose 1,6 biphosphatase, and thus high [ATP] would correspond to high activity of fructose 1,6 biphosphatase.*
*Which of the following statements are true? (select all that apply)* i. PFK1 must have more than one Fructose-6-Phosphate (F6P) binding site. ii. The activity of PFK1 shows Michaelismenten kinetics iii. F6P is a homotropic effector of PFK1 iv. F6P is a heterotropic effector of PFK1 v. F6P is an inhibitor of PFK1
i. PFK1 must have more than one Fructose-6-Phosphate (F6P) binding site. iii. F6P is a homotropic effector of PFK1 *(homotropic effector = the allosteric regulator and the substrate are the same molecule)*
*Pyruvate kinase isoform M2 (PKM2) is an enzyme that catalyzes the final step of glycolysis in cancer cells. A feature distinguishing PKM2 from PKM1 is that PKM2 is regulated by fructose-1,6-bisphosphate. What type of regulation is the regulation of PKM2 by F1,6BP? Select all that apply.* i. allostery ii. feedback inhibition iii. isozyme iv. covalent modification v. feed forward activation vi. proteolytic cleavage
i. allostery v. feed forward activation *(an early product in the pathway activates (or catalyzes) future reactions. The concentration of an earlier reactant not involved in the continual chemical reactions of the pathway will help to catalyze final reactions down the pathway)*
What is the effect of an enzyme/catalyst on a reaction? Select one answer. i. makes the reaction thermodynamically favorable ii. increases the rate of reaction iii. decreases the rate of reaction iv. increases the activation energy v. changes the energy of the product
ii. increases the rate of reaction
