Exam 2 Review Flashcards
What is the basic principle of catalysis?
Stabilizing transition states
What are the 6 classes of enzymes (based on the reactions they catalyze)?
- Oxidoreductases
- Transferases
- Hydrolases
- Lysases
- Isomerases
- Ligases
What function do oxidoreductases have?
They perform redox reactions, which involve the transfer of electrons
Ex: Oxidases, peroxidases, reductases …
What is the characteristic feature of all hydrolases?
They all use H20 molecules to assist with the catalysis
What is the difference between a Lyases and a Ligase?
Lyases do not need energy, whereas ligases use triphosphate molecules (XTP, i.e. ATP) to help drive the reaction
What group of enzymes does ATP synthase fall into?
ATP synthase is a hydrolase because it uses an H20 molecule to help form ATP molecules
What is Gibbs free energy?
The energy available to do work (drive chemical reactions)
What is standard free energy?
The free energy at specific standard conditions:
25 degrees C, 1atm, pH=7
Do enzymes affect the delta G of a reaction?
No, enzymes only change the energy that is required to initiate the conversion of reactants into products (rate of reaction)
What is an exergonic reaction?
Exergonic means that the reaction is spontaneous. The reaction proceeds in the direction that leads to a loss of free energy “Downhill”
Can enzymes catalyze nonspontaneous reactions?
No. Enzymes do not change delta G, they only change the rate of the reaction
How can you make a nonfavorable reaction happen?
You can couple it with a favorable reaction with a larger deltaG
ex: ATP hydrolysis
What does a negative delta G signify?
A spontaneous reaction
What is the law of mass action?
The equilibriam constant can becalculated in the follwing way:
For Rxn: A + B > C + D
K = [C][D] / [A][B]
What is the relationship between delta Go and K?
Go = - R T ln(K)
T = temperature in kelvin
R= 8.314 J/(mol*K)
K = equilibrium constant
How do you calculate the delta G of a reaction in non-equilibrium conditions?
ΔG = ΔGo + R*T* ln ( [C] [D] / [A][B] )
where A, B, C, D are the molar concentrations of the products and reactants
Is ΔGo’ positive or negative if there is more products than educts?
ΔGo’ is negative
Describe the ΔGo of pacemaker reactions, such as the conversion of ATP to ADP + Pi
Pacemaker reactions have very large negative ΔGo’ values
How can the spontaneity of a reaction with a small ΔGo’ be changed?
By changing the concentrations of reactants or products
What is activation energy, and how do enzymes affect it?
Activation energy is the free energy difference between the substrate and the transition state. Enzymes lower activation energy, allowing more molecules to have enough energy to reach the transition state.
Describe the bonding between substrate and enzyme
Multiple weak interactions bind the substrate to the enzyme including: electrostatic forces, H-bonds, and vanderwaals forces
How does the enzyme promote the formation of the transition state?
It promotes the substrate(s) to be positioned in favorable orientations, making formation/breaking of appropriate bonds more likely to occur
Why is it not strictly correct to say “a catalyst reduces the activation energy of a reaction”?
Catalysts allow a completely different reaction to take place that has the same substrate and produces. This reaction has a lower activation energy than the uncatalyzed reaction
What does the fact that enzymes have a Vmax suggest about how substrates and enzymes interact?
The existance of a maximal velocity suggests that substrates and enzymes form discrete ES complexes. The vmax occurs when all enzymes are bound to substrates.
Describe the active site of a typical enzyme
- 3D cleft formed from tertiary protein structure
- precise arrangement and nature of molecules determine the substrate specificity
- microenvironment that promotes the reaction
- small volume relative to entire enzyme
- mutliple weak interactions form ES
What is the difference between the “lock & key” and “induced fit” models?
In the lock and key model, only specific substrates can bind to an enzyme because of their inherent shape
In the induced fit model, the enzymes are flexible and can conform to fit to several differently shaped substrates. (catching a baseball analogy)
What is binding energy?
The free energy that is released on binding.
This energy is maximized when the correct substrate binds to an enzyme because it has the maximum possible interactions within the active site.
What metal ion is bound to carbonic anhydrase?
Zn2+ is bound to three histidine rings in the interior B-sheets of the carbonic anhydrase enzyme
What is the unit for the rate constant of a first order reaction?
1/s
What is the equation for the velocity of a first order reaction?
v = k*[A]
where v=velocity
k=rate constant
[A] = concentration of substrate A
What is the equaltion for the velocity of a second order reaction?
v = k*[A]*[B]
where k = rate constant
[A], [B] = concentrations of substrates
What is the steady state assumption?
The concentrations of intermediates ([ES]) stay the same even if the concentrations of starting materials and products are changing
What quantity is varied in order to measure the initial velocity of an enzyme?
[S] is varied while [E] is held constant.
As [S] is increased, Vo increases until it the enzymes are saturated at vmax
What quantity tells us about the formation of the ES complex?
Vo: initial velocity
What is Km?
Km is the Michaelis constant and equals the substrate concentaration at which the reaction rate is half of its maximal value
When [S] <<< Km, what does Vo equal?
Vo = (Vmax/Km) * [S], if [S] <<< Km
When is the reaction velocity substrate dependent?
At low substrate concentrations, the velocity curve has a large slope, making it substrate dependent. At high [S] the enzymes saturate and the velocity is independent of [S]
What can k2 also be called?
The catalytic constant or turnover number
What is the equation for vmax?
Vmax is at high substrate concentrations, so all E will be bound in ES form.
vmax = Kcat * [Et]
where Et is total enzyme concentration
What are the two partial reactions involved in Michaelis-Menten kinetics?
1) formation and decay of the enzyme-substrate complex (km)
2) formation of the product (kcat)
What is the Michaelis-Menten equation?
v = (kcat*[Et] * [S]) / (km + [S])
What are the axes on a Lineweaver-Burk plot?
x-axis: 1/[S]
y-axis: 1/v
What is the significance of the intercepts on a lineweaver-burk plot?
The y-intercept = 1/Vmax
The x-intercept = -1/Km
What does the Km value say about the affinity of an enzyme for a substrate?
A low Km signifies a high affinity for the substrate
*half of the maximal velocity is reached at a lower [S]
Does lower Km mean a higher vmax?
No. Km and Vmax are independent
How is the rate constant of an enzyme defined?
rate constant = kcat/ Km
Can an enzyme have more than one rate constant?
Yes. Different an enzyme that binds to different substrates like chymotrypsin has a different rate constant for each ES complex.
What are the two types of enzyme reactions with multiple substrates?
Sequential and Double Displacement (ping-pong)
Explain what happens during a double-displacement “Ping-pong reaction”.
A substituted enzyme intermediate is formed when the first substrate interacts with the enzyme.
This Enzyme intermediate is then able to interact with a second substrate to form the product.
It is called ping-pong because the substrates appear to bounce off of the enzyme
Explain the binding energy curve of an allosteric enzyme.
The binding energy peaks at [A]50, where the velocity is equal to half Vmax
It then decreases as [A] increases
This is due to the cooperative binding. Once one substrate binds, the enzyme changes into a complex that has a higher affinity for the substrate.
What are additional factors that affect the activity of an enzyme?
pH and temperature change the conformation of an enzyme
these can be used to regulate enzyme activity
What is the critical temperature that most enzymes denature at?
42 degrees celcius
What are isozymes?
Enzymes that differ in amino acid sequence, but catalyze the same chemical reaction
They have different Km and Vmax
How if the fractional saturation of an enzyme calculated?
fes
fes = V/Vmax = [S] / ([S] +Km)
Which type of inhibition is characterized by rapid dissociation of the enzyme-inhibitor complex?
Reversible inhibition (competitive)
Describe competitive inhibition
Lowers the rate of catalysis by reducing the proportion of enzyme molecules bound to substrate
Km increases
Vmax is unchanged
Inhibitors can be substrate or transition state analogs
How does the slope of the L-B plot change with the addition of a competitive inhibitor?
The slope increases and the y intercept (and thus Vmax) stays the same
Ibuprophen is an example of which type of inhibitor?
Competitive inhibitor
Describe uncompetitive inhibition
The inhibitor only binds to the ES complex, preventing the S from being released
Km and Vmax are reduced
The L-B plot is shifted left (higher intercepts)
Describe noncompetitive inhibition
The inhibitor can bind to E or ES
Vmax is reduced, but Km stays the same
What are suicide inhibitors?
Starts out like competitive inhibition with a substrate analog, but then a covalent bond is formed between the inhibitor and the active site of the enzyme.
This appears like non-competitive inhibition.
Example: Penicillin
What are the 4 catalytic strategies?
- Covalent catalysis
- General acid-base catalysis
- Catalysis by approximation
- Metal Ion catalysis
Describe how proteases like chymotrpsin work
Proteases use covalent catalysis to destabilizze and break a peptide bond through hydrolysis.
The serine OH group is able to act as a nucleophile and attack the carbonyl of the target peptide bond (Acylation)
Then H2O deacylates the group leaving behind the enzyme and cleaved peptide
What is the function of the oxyanion hole?
It stabilizes the tetrahedral intermediate during covalent catalysis
Compare and contrast the role that Mg2+ plays in ATP hydrolysis to the role it plays in restriction endonucleases
Mg2+ plays similar roles in both, but it is included as part of the ATP substrate in ATP hydrolysis, whereas it is part of the restriction endonuclease enzyme itself
Describe the power stroke of myosin
The hing region of the myosin undergoes a large conformational change which allows for the myosin head to be displaced, thus leading to mechanical contraction
What does transcriptional control of enzymes regulate?
The number/concentration of enzymes
ex: transcription factors
Describe interconversion in terms of enzyme regulation
Second messenger systems are used in order to activate/de-activate enzymes
Which step of a metabolic pathway do key enzymes catalyze?
The slowest step
What is a zymogen?
An inactive form of an enzyme. This can be cleaved in order to activate it.
What is the function of aspartate carbamoyl-transferase (ATCase)?
It catalyzes the first step in the syntesis of pyrimidines such as CTP
Describe how ATCase is regulated
CTP inhibits the enzyme via negative feedback
ATP activates the enzyme
Describe the structure of ATCase
It is made up of 6 dimers that are arranged into 2 groups of 3 stacked on top of one another. Each dimer has a catalytic and regulator end.
ATP and CTP can both bind to the regulatory subunit
2 conformations: T and R state
Describe the T and R states of ATCase
The R conformation is more active than the T state
CTP binding favors the T state, and substrate binding favors the R state.
In the R state, the regulatory subunits rotate, moving the catalytic subunits apart
Which direction does the binding curve shift in the presence of a negative regulator?
The sigmoidal curve is shifted right for negative regulators
Ex: CTP affecting ATCase
What is L in the context of allosteric regulators?
L is the ratio of molecules in T state to R state
L= T/R
Describe the Hill coefficient
The hill coefficient is a measure of the cooperativity of oxygen binding
Does the conversion of ATP to ADP and Pi have a positive or negative Gibbs Free Energy?
Very Negative
-30 kJ/mol
What role does the P-loop play for ATP Hydrolysis?
The P-loop is lowered and is able to stabilize the ATP within the binding sites
Can a monomeric protein display cooperative binding?
No. Cooperative modulation is only possible in multimeric protein complexes. When a substrate binds to one subunit, the binding affinity increases for the other subunits
What molecule provides the phosphate for phosphorylation reactions?
ATP
What are the only amino acids that can be phosphorylated?
Serine, Threonine and Tyrosice
These amino acids have hydroxyl groups capable of binding covalently to phosphate groups
What role do kinases and phosphatases play?
Kinases add a phosphate group
Phosphatases remove a phosphate group
How is Protein Kinase A regulated?
Via covalent modification.
Regulatory chains have a sequence that is a substrate analog which occupies the active site (competitive inhibition)
cAMP can bind to the regulator subunits allowing them to be released from the catalytic subunit leading to activation of PKA
Where is inactive PKA found in the cell?
inactive PKA is found in the cytosol of the cell
Active PKA enters the nucleus because of a nuclear localization sequence (NLS) which is recognized by nuclear transport proteins
What molecule is a general activator for most proteases?
Trypsin cleaves many proteases from their zymogen form to the active form
What is known as the master regulatory step for proteases?
The transmembrane protein enteropeptidase catalyzes the conversion from trypsinogen to trypsin
What are the two major parts of the blood clotting cascade?
The intrinsic pathway and the extrinsic pathway
What molecule is formed via both the intrinsic and extrinsic blood clotting pathways and plays a major role in the formation of clots?
Clotting factor Xa allows for the activation of thrombin, which catalyzes the conversion of prothrombin to thrombin
What happens in hemophiliacs?
They are unable to form blood clots properly, which leads to excessive bleeding. They cannot properly form Factor Xa
How is the clotting cascade deactivated?
Plasminogen is converted to plasmin which is able to dissolve the fibrin thrombus.
Tissue plasminogen activator (TPA) catalyzes the conversion of plasminogen to plasmin and is located within the fibrin thrombus
What molecule is commonly referred to as blood sugar?
Glucose
What is the major glucose storage molecule and where is it found in the body?
Glycogen is found in the liver and muscles
What is the glycocalyx?
The “sugar forest” on the exterior surface of cell membranes.
Useful for the identification of cells
What role do carbohydrates play in terms of hydration of cells?
Carbs can bind water to prevent cells from drying out
What are isomers?
Molecules with the same molecular formula but different structures
What are constitutional isomers?
They differ in the order of attachment of atoms
ex: aldehydes and ketones
What are stereoisomers?
Atoms that are connected in the same order but differ in spatial arrangement
What are enantiomers?
Nonsuperimposable mirro images
ex: D-glyceraldehyde, L-glyceraldehyde
What are diastereoisomers?
Isomers that are not mirror images
ex: D-altrose and D-glucose
What are epimers?
Molecules that differ at one of serveral asymmetric carbon atoms
What are anomers?
Isomers that differ at a new asymmetric carbon atom formed on ring closure
alpha-Glucose and beta-Glucose
the direction of the hydroxy group determines which anomer exists
In what form is sucrose found?
It is found as a dimer where the anomeric carbons are bound together.
What are the differences between Pyranose and Furanose?
Pyranoses are 6 membered rings formed from aldehydes. Furanoses are 5 C rings formed from ketones
What is the difference between ribose and deoxyribose?
Deoxyribose does not have a 2’ hydroxyl group
Describe glycosidic bonds
Covalent bonds between 2 carbohydrate models.
They can either be O-glycosidic bonds attached to an oxygen or N-glycosidic bonds connected through a N that has replaced an OH group
What is advantageous about converting glucose to G6P within the cell?
The phosphorylation of glucose maintains the glucose concentration gradients.
Additionally, the phosphate group destabilizes the ring structure which makes it easier to break down in later glycolytic steps
What are the structural differences between Glycogen and Starch?
Glycogen is a branched glucose polymer found in animal cells. 1-4 glycosidic bonds create the chains, and 1-6 glycosidic bonds create the branches
Starches can be branched or unbranched glucose polymers that are found in plant cells
What cells in the body rely exclusively on sugar for energy?
Red blood cells and brain cells
Describe the structure of cellulose
Cellulose is combosed of many chains of glucose connected via 1-4 glycosidic bonds. Hydrogen bonds form between strands to make a highly stable network.
