Exam 2 Flashcards
(118 cards)
1
Q
Definition: Heme
A
Cofactors in many different proteins, the most important of which are hemoglobin, myoglobin and cytochromes
2
Q
Definition: Cytochromes
A
Electron transfer proteins involved in metabolism
3
Q
Definition: Porphyrin
A
- The ring that surround the iron ion in hemoglobin
- May be covalently attached via chemical groups to the protein backbone
4
Q
Definition: Allostery
A
Proteins that have multiple binding sites (either the
same or different), in which the binding of a ligand at the first site (allosteric site) causes a conformational change that alters the affinity of binding at the second site.
5
Q
Definition: Positive cooperativity / Cooperative binding
A
In allostery, if the binding affinity at the second site is enhanced by ligand binding at the first site
6
Q
Definition: Negativity cooperativity
A
iIn allostery, if the binding affinity at the second site is decreased by ligand binding at the first site
7
Q
Myoglobin
A
-Consists of a single globin chain, and therefore has a typical hyperbolic binding curve
-At partial pressures of oxygen typically found
in both the lungs and tissues, it remains highly saturated with oxygen, making it an effective oxygen storage molecule, particularly in muscle
-If the oxygen level falls below the normal levels required by the tissues, then myoglobin begins releasing oxygen to supply the cell with its needs
8
Q
Hemoglobin (Hb)
A
-Exhibits positive cooperativity
-At the lungs, Hb is saturated with oxygen (it “picks up” oxygen) but at the levels normally found in tissues, it releases a significant amount (the lower the tissues’
levels, the more it releases) thus making it a good oxygen transport molecule
9
Q
In what two states might you find the hemoglobin subunits?
A
i) Tense (T) - It is harder for O2 to bind
ii) Relaxed (R) - Facilitates O2 binding
10
Q
True or false: Binding of the first molecule of O2 to
deoxygenated hemoglobin causes conformational changes that make binding of O2 to the other subunits easier
A
True
11
Q
What two models of cooperative binding exist for hemoglobin? Does it exhibit behavior consistent with both models or just one?
A
i) “All-or-Nothing” Model - The binding of the first O2 to a hemoglobin subunit immediately makes all other subunits change conformation to the R state
ii) Sequential Model - The binding of the first O2 to a hemoglobin subunit then changes the next subunit into R form. After the second subunit binds O2, it changes the next subunit to R form, and so on, until all subunits have changed to R form and bound O2
—Hemoglobin has behavior consistent with both models!!
12
Q
Is DeoxyHb in the T state or R state? What state is the heme in?
A
DeoxyHB is in the T state. The heme is in the Fe2+ state, not oxygenated at all
13
Q
Is OxyHb in the T state or R state? What state is the heme in?
A
OxyHb is in the R state. The heme is fully oxygenated with all subunits bound to Fe2+
14
Q
True or false: In the absence of O2, the R state is more stable
A
False - In the absence of O2, the T state is more stable. The R state is more stable in the presence of O2.
15
Q
True or false: O2 binding to hemoglobin is dependent on only on pH
A
False - O2 binding to hemoglobin is dependent on both pH AND CO2
16
Q
Why might hemoglobin want to have a lower binding affinity for O2? (The Bohr Effect)
A
In tissues, where the pH is relatively low and the CO2 concentration is high (produced by biochemical reactions in the tissues), hemoglobin “drops” O2, which is released to the tissues. Hemoglobin then actually binds some of the excess CO2 and also becomes protonated (binds H+).
—Therefore, hemoglobin can act as a removal system for unwanted things and then carry O2 back to the tissues on the return trip
17
Q
What conditions of pH and CO2 causes hemoglobin to bind to O2 with less affinity?
A
A lower pH and/or increased CO2 causes lower binding affinity (such as in tissues)
18
Q
Definition: Catalytic power
A
- The rate of the catalyzed reaction relative to the rate of the uncatalyzed reaction
- Catalytic power of enzymes is usually in the range of 10^6 to 10^14
19
Q
Definition: Ligand
A
The molecule that binds in a reaction
20
Q
Definition: Desolvation
A
- An energy penalty in removing bound water molecules from the binding site and the ligand
- This occurs as a result of the formation of many non-covalent interactions during ligand binding
21
Q
Why are chemical interactions dependent upon concentration?
A
The more molecules there are, the more likely that two of them will bump into each other in a productive manner
22
Q
Definition: Lock-and-Key hypothesis
A
- The complementarity of the binding surfaces leads to enzymes, receptors and antibodies being very specific for certain ligands
- Essentially, certain enzymes are a perfect fit for certain, specific molecules
23
Q
Definition: Induced fit hypothesis
A
- Upon initial binding, enzymes undergo some level of conformational change to optimize the interactions
- Essentially, the enzymes and molecules may not be a PERFECT fit for each other, so they move slightly to improve their binding capabilities
24
Q
True or false: Enzymes speed up the forward AND backward reactions
A
True - Because of this, equilibrium DOES NOT CHANGE! The forward and backward reactions still take place at the same ratio, just faster in total
25
True or false: The larger the Keq (equilibrium constant), the more positive the delta G
False - As Keq becomes larger, delta G becomes more negative, making it a more spontaneous reaction
26
What is the equation for the equilibrium constant (Keq)?
Keq = [LP] / [L][P]
--> [LP] is the concentration of products of bound ligand to protein, where [L] and [P] separate are the concentrations of reactant before binding
27
True or false: The larger the Ka, the stronger the binding affinity of the ligand to the protein
True - The opposite is true for Kd, as this is the reciprocal of Ka (for Kd, the smaller it is, the stronger the binding affinity)
28
True or false: The Keq and the Ka can be represented by the same equation
True ; Keq = [LP] / [L][P] AND Ka = [LP] / [L][P]
29
How can you evaluate the specificity of a receptor if there are two similar ligands present?
- Compare the individual dissociation constants, Kd, for each of the two ligands
- The receptor is said to be more specific for the ligand with the tighter binding (smaller Kd)
30
What is the hill coefficient (nH) a measure of? Explain what nH > 1, = 1, and < 1 means.
It is a measure of cooperativity
- nH > 1 is positive cooperativity
- nH < 1 is negative cooperativity
- nH = 1 there is no cooperativity
31
What does kcat represent?
- The turnover rate of an enzyme
| - Essentially, how fast enzyme can turn substrate into product
32
True or false: The steady state of an enzyme-catalyzed reaction is reached when the concentration of the enzyme-substrate complex is constant over time
True
33
True or false: The steady state of an enzyme-catalyzed reaction is reached when the rate of appearance of product over time is constant
False - The steady state of an enzyme catalyzed reaction is reached when the CONCENTRATION (not the formation!!) of the enzyme-substrate complex is constant over time
34
What is the Michaelis-Menten equation?
Vo = Vmax*[S] / (Km + [S])
35
What is the michaelis-menten constant (Km) a measure of?
- It is a measure of the affinity of the enzyme for the substrate
- As Km becomes smaller, the affinity becomes greater
36
Where is Km on a plot?
It is the concentration of substrate ([S]) at which the initial velocity (Vo) = 0.5*Vmax
-Essentially, the X value that matches the Y value of 0.5*Vmax
37
If, in a reaction, Kcat decreases and Km remains neutral, what is occurring?
What this likely means is that it has something to do with the K2 rate constant. It is changing the rate of substrate into product after binding occurs (slows down catalytic step) ; It does not play a role in the initial binding.
38
If, in a reaction, Kcat remains neutral and Km increases, what is occurring?
The increase in Km is now worse at binding (like Kd, larger numbers mean less affinity). This amino acid helps the substrate bind to the enzyme (1st step). Has nothing to do with the ability of the enzyme to turn bound substrate into product (which Kcat is involved in)
39
What is the efficiency value equation for Michaelis-Menten reactions?
Efficiency = Vmax / Km
40
How does competitive inhibition work?
- A competitive inhibitor competes for the same binding site as substrate; the binding is reversible.
- A competitive inhibitor often chemically resembles substrate, but the enzyme cannot catalyze its conversion to anything else
41
True or false: The best competitive inhibitors are analogs of the transition state
True - The binding sites of enzymes are optimized for binding the transition state (thus lowering the energy required to cross the transition state barrier). When substrate binds, the enzyme is set up to “encourage” the substrate to optimize its interactions with the enzyme even more by adopting the structure of the transition state. Thus drugs that closely resemble the transition state will have higher binding affinities to the enzyme than pure substrate analogs.
42
What does alpha equal in inhibition equations?
alpha = 1 + ([I] / KI)
43
How does a competitive inhibitor affect Km and Vmax?
- The presence of a competitive inhibitor increases the experimentally measured Km, but does not affect the Vmax
- This occurs because when [S] >> [I], all the active sites will be occupied by substrate, not inhibitor, thus leaving Vmax unaltered. The Km will not be the “true” Km for substrate, but an “apparent” Kmapp
- Will have the same y-int but a different x-int
44
How do pure noncompetitive inhibitors work?
- They reversibly bind at another site on the enzyme away from the substrate binding site
- The binding of the inhibitor does not alter the binding affinity of the substrate for the enzyme, i.e. no inhibitor induced conformation change that affects the substrate binding site. Therefore, substrate can still bind to the enzyme in the presence of the inhibitor, but the reaction will not proceed to product with inhibitor bound.
45
How does a pure noncompetitive inhibitor affect Km and Vmax?
- Km is unaffected, and Vmax is decreased
- The inhibitor is not binding to the same site as the substrate, and does not affect Km. The inhibitor binds to both the free enzyme and the ES complex, and its binding does not affect binding of the substrate to the enzyme
- However, the Vmax for the enzyme will not be the “true” Vmax, but an “apparent” Vmaxapp. The inhibitor I is taking some proportion of E and EI “out of commission,” so the effective turnover rate (kcat) at high [S] will be decreased relative to the conditions with no inhibitor
- Will have the same X-int, but the maximum y value will be different
46
How does uncompetitive inhibition work?
- An uncompetitive inhibitor can only bind to the ES complex; The binding is reversible
- This type of inhibition occurs most commonly in multisubstrate reactions, where the addition of substrates is controlled sequentially (ordered) by a conformational change induced by binding of the first substrate that enables binding of the second substrate at a separate site
47
True or false: An uncompetitive inhibitor with respect to the first substrate is a competitive inhibitor with respect to the second substrate
True
48
How does uncompetitive inhibition affect Km and Vmax?
- It lowers both Km and Vmax
| - The graph is parallel lines (different X int AND different Y int)
49
How does a mixed noncompetitive inhibitor work?
- Reversibly binds at another site on the enzyme away from the substrate binding site
- The binding of the inhibitor does alter the binding affinity of the substrate for the enzyme, i.e. an inhibitor induced conformation change that affects the substrate binding site
- The binding of substrate alters the binding affinity of the inhibitor for the enzyme, i.e. there is a substrate induced conformational change that affects the inhibitor binding site.
50
How does a mixed noncompetitive inhibitor affect the Km and Vmax?
It affects both Km and Vmax. The 1/[S] v. 1/Vo graph looks like the competitive inhibitor graph, except that both the X-int and Y-int are different, rather than just the X-int as in competitive
51
What does suicide inhibition do?
- It is an irreversible inhibition type which effectively kills the enzyme to which it attaches
- It is irreversible because it is a covalent attachment-- all other inhibitors are non-covalent
52
What does alpha equal in a pure noncompetitive inhibitor?
alpha = Vmax / Vmaxapp
53
What does the plot of a Sequential Bi Bi reaction look like?
It is three lines which have one x-int but have different slopes (diff. y-int). The constant increases as the slope gets smaller.
54
What does the plot of a Ping-Pong Bi Bi reaction look like?
Three parallel lines (same slope, different x- and y-int). The constant increases as the slope gets smaller.
55
What seven amino acid side chains act as general acid-base catalysts?
i) Asp
ii) Glu
iii) His
iv) Cys
v) Tyr
vi) Lys
vii) Arg
56
What is the main factor in determining if a side chain is acting as a base or an acid?
- One acting as a base will contain either a negatively charged Oxygen or a neutral Nitrogen
- One acting as an acid will contain a neutral Oxygen bound to a hydrogen, or a positively charged Nitrogen
- --> The nitrogens may or may not be attached to a hydrogen regardless of their charge
57
How is rate acceleration achieved in covalent catalysis?
- It is achieved through formation of a transient covalent bond between substrate and enzyme
- The bond formed many times activates the substrate for subsequent reaction (i.e. the covalent bond directs the path of the reaction)
- Subsequent reactions release product and regenerate enzyme
58
True or false: Highly polar groups make good covalent catalysts
True
59
Must a catalytic group involved in covalent catalysis be a good nucleophile, a good leaving group, neither, or both?
It must be both. It must be a good nucleophile (to form the covalent bond) and a good leaving group (to break the bond)
60
What six amino acid side chains can be involved in covalent catalysis?
Lys, His, Cys, Asp, Glu and Ser side-chains can all be involved in covalent catalysis
61
List three common metalloenzymes
-Metalloenzymes contain tightly bound metals, most usually transition metals, such as Fe, Cu, Zn
62
List three common metal-activated enzymes
-Metal-activated enzymes loosely bind metals, most usually alkali or alkaline earth metals, such as Na, Ca, Mg. These metals often come associated with a substrat
63
What is the point of having metal involved in catalysis?
- Metals can promote water ionization to yield OH- ions that participate in nucleophilic attack
64
Why might the kinetic values (Kx values) be similar for both a forward and reverse reaction?
If the reaction needs to be readily reversible, it will have similar kinetic values for both forward and reverse
65
What does the glycolytic enzyme enolase catalyze?
- It catalyzes reversible dehydration of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate
- --Takes an H2O off the beta carbon
66
What two residues take part in the enolase reaction?
i) Lys 345 - Stabilized by Mg2+
| ii) Glu 211
67
What is electrostatic catalysis?
- This is the stabilization of enzyme bound intermediates that develop a charge
- Generally this involves oppositely charged amino acids, or hydrogen bonds
- The most well-studied example is known as the oxyanion hole
68
What is an oxyanion hole?
- An oxyanion hole involves the stabilization of a negatively charged covalent catalytic intermediate by a positively charged amino acid, or by hydrogens containing a partial positive charge due to their covalent bonding to an electronegative atom (i.e. hydrogen bonding)
- Is a well-studied example of electrostatic catalysis
69
In terms of position and orientation, how do enzymes work on a substrate?
-Enzymes immobilize and correctly orient the substrate
70
What do proteases do?
-Proteases catalyze the hydrolysis of polypeptide backbones to yield smaller peptides
71
Describe serine proteases`
- Serine proteases have a peculiarly reactive Ser residue that is essential for catalytic activity, and all utilize the same catalytic mechanism
- The best studied serine proteases are the animal digestive enzymes
72
Where does Trypsin (a serine protease) cleave?
-Cleaves C terminal side of Arg and Lys
73
Where does chymotrypsin (a serine protease) cleave?
-Cleaves C terminal side of Phenylalanine, Tyrosine, and Tryptophan
74
What does elastase (a serine protease) cleave?
-Cleaves smaller, non-polar amino acids Alanine, Glycine, Serine, and Valine
75
What three residues make up the catalytic triad of chymotrypsin? What is this triad bound by?
i) Ser 195
ii) His 57
iii) Asp 102
-Bound by hydrogen bonds
76
In the first step of serine protease's mechanism, where does the enzyme bind to the substrate? (chymotrypsin)
-In a hyrophobic pocket
77
Describe the second step of serine protease's mechanism (chymotrypsin)
- Nucleophilic attack by Ser OH
- --a) His57 deprotonates Ser195 OH via general base catalysis
- --b.) Ser O- attacks carbonyl carbon (nucleophilic) via proximity and orientation
- --c.) Forming tetrahedral intermediate 1 via covalent catalysis
78
Describe the third step of serine protease's mechanism (chymotrypsin)
- Protonation of peptide amino group
- --a.) His 57 protonates peptide amino group via general acid catalysis
- --b.) Peptide bond is cleaved
- --c.) N-terminal fragment still attached to Ser 195 (via acylenzyme intermediate)
- --d.) Neutron diffraction shows that the proton between His57 and Asp102 remains with His (via electrostatic catalysis
79
Describe the fourth step of serine protease's mechanism (chymotrypsin)
- Deprotonation of H2O and attack carbonyl carbon (H2O is second substrate)
- --a.) His57 acts as a base to abstract proton from H2O – general base catalysis. (note: pH must be high enough so that His57 isn’t protonated!)
- --b.) Resulting hydroxyl ion attacks carbonyl forming tetrahedral intermediate 2
80
Describe the fifth and sixth steps of serine protease's mechanism (chymotrypsin)
- Cleavage of Ser O-peptide bond
- --a.) His57 donates a proton to Ser O via general acid catalysis
- --b.) Cleaving acyl-enzyme intermediate
- --c.) Releasing N-terminal fragment
81
True or false: The oxyanion hole enables preferential binding to the transition state in serine protease
True
82
What occurs during the final step of serine protease's mechanism? (chymotrypsin)
-Diffusion of second product (new C-terminus) from the active site to regenerate free enzyme
83
What enzyme breaks down NAG and NAM in bacterial cell walls?
Lysozyme (Hen Egg White Lysozyme, to be specific (HEWL))
84
What are the two residues involved in lysosyme?
- i) Glu 35
| - ii) Asp 52
85
What was the difference between the Phillips and Wither's mechanisms for lysosyme?
- Difference is the intermediates: carbocation intermediate (Phillips) versus covalent intermediate (Withers)
- For Phillips mechanism, Asp52 with negatively charged oxygen stabilizes carbocation (electrostatic catalysis)
- For Withers mechanism, the covalent bond formed between Asp52 stabilizes the intermediate (covalent catalysis)
86
Is lysozyme sequential or ping pong?
Ping pong (bi bi also)
87
What does HIV protease do?
Cleaves HIV polypeptides synthesized in the infected host which then become functional proteins used for making more of the virus
88
What side chains are involved in HIV protease?
- Aspartyl protease: two active site Asp25 amino acids
| - No covalent catalysis (unlike Serine proteases or Cysteine proteases)
89
True or false: Protease recognizes and cleaves peptides between Pro-Pro most efficiently
False: Protease recognizes and cleaves peptides between Phe-Pro most efficiently, NOT Pro-Pro
90
True or false: The tetrahedral intermediate in HIV protease is stabilized by covalent bonds
False: The tertahedral intermediate is stabilized by hyrodgen bonds
91
How does the inhibitor of HIV protease work?
The presence of an –OH group in the inhibitor which mimics the negatively charged (and stabilized) oxygen in the tetrahedral intermediate binds extremely strongly
92
What is the point of a regulatory enzyme?
Regulation keeps un-needed products from being made and wasting the energy store of the cell
93
True or false: The first enzyme in a sequence is commonly the regulatory enzyme
True
94
Describe slow enzymatic response. What three kinds of enzyme regulation work this way?
- Enzymes can be regulated through their abundance with no change in activity. Longer lasting than fast response
- Changes in abundance levels of enzymes are controlled by gene regulation, protein synthesis and degradation
95
Describe fast enzymatic response. What three kinds of enzyme regulation work this way?
- Enzymes can be regulated through a change in their specific activity, with no change in their abundance
- Changes in specific activity involve allosteric regulation, covalent modification and zymogen activatio
96
Definition: Allosteric Activators
- Enhance catalytic activity
- Decrease the Km and/or increase the Vmax -Molecules that decrease the catalytic activity are known as allosteric inhibitors
97
Definition: Allosteric inhibitors
- Decrease catalytic activity
- Increase the Km and/or decrease the Vmax
- Binding of the allosteric molecule leads to a conformational change that affects the active site
98
True or false: Allosteric enzymes are generally large, single unit proteins
False: Allosteric enzymes are generally large, MULTISUBUNIT proteins
99
If 2 ligands bind preferentially to the same conformation of the protein, one enhancing the binding of the other, then (activation/inhibition) occurs
Activation
100
If 2 ligands bind to different conformations of the protein, one reducing binding of the other through a larger conformational switch, then (activation/inhibition) occurs
Inhibition
101
Describe feedback inhibition
Feedback inhibition occurs when a late product from a multienzyme pathway binds to an allosteric site (also called a regulatory site) on an early acting enzyme in the pathway, and inhibits its action. This is negative regulation.
102
Describe positive inhibition
The product from one pathway stimulates an early enzyme in a different pathway.
103
Is feedback inhibition reversible or irreversible?
Fully reversible
104
What is the most common activated carrier molecule?
ATP
105
Describe the regulation of glycolysis
-Citrate and ATP inhibit phosphofructokinase-1, while AMP (indicating the cell is in a low energy state) activates the enzyme.
106
What is a zymogen?
The inactive form of an enzyme that requires additional processing by enzymes at their final cellular destination to become active (either the proteolytic cleavage of the polypeptide chain, a post-translational modification, or cofactor insertion)
107
What does a proteasome do? Also, describe its basic structure
- Proteasomes chew up big proteins and spit out smaller ones
| - They are a little tube with a cap at one end. The cap recognizes proteins targeted for degradation
108
What is the point of ubituitin?
It targets proteins to proteasomes for degradation
109
What does E1 do in ubiquitination?
E1 activates ubiquitin by forming a highly reactive thioester bond with the C-terminus and an E1 Cys residue. This is an ATP dependent process. This thioester bond is highly reactive to the nucleophilic amine group of lysine.
110
What does E3 do in ubiquitination?
The E3 recognizes a specific degradation signal on the target protein, enabling E2 to transfer its ubiquitin to a Lys on the target
111
What is the point of phosphorylation?
It is used to reversibly regulate protein function through conformational changes
112
What is the name of an enzyme which phosphorylates?
Protein kinases
113
What is the name of an enzyme which dephosphorylated?
Protein phosphatases
114
Why is ATP stable in aqueous environments?
ATP is actually very stable in aqueous solutions (i.e. within the cell) due to high activation energy for hydrolysis
115
What is phosphoenol pyruvate catalyzed by?
Enolase
116
What is phosphoenol pyruvate hydrolyzed to?
Phosphoenol pyruvate (catalyzed by enolase) is hydrolyzed to pyruvate (product of glycolysis). The tautomerization of the product makes it energetically favorable
117
True or false: ATP drives a reaction by itself
False: In reality, the hydrolysis of ATP generates heat, but doesn’t help to “drive” a reaction
-Instead, ATP-driven biochemical reactions are many times two-step transfer reactions
118
Why is a phosphaste bond called 'high energy'?
- The free energy release from P-O bond breaking is due to the lower free energy of the products relative to the reactants
- So when you hear “high energy phosphate compound” etc. think of these as being involved in reactions with large, negative delta G values
