1
Q
What are the two types of “globin” and what do they do?
A
Myoglobin and hemoglobin – bind O2 at iron in heme
2
Q
How does hemoglobin bind to O2?
A
Hemoglobin binds O2 cooperatively
3
Q
What is the Bohr effect?
A
H+ and carbon dioxide stabilize the T state once oxygen gas is released
4
Q
What’s the significance in mutations in the hemoglobin gene?
A
It serves as a biochemical basic for the symptoms of Sickle-cell anemia
5
Q
What are the differences between hemoglobin and myoglobin?
A
- Hemoglobin - oxygen transport through blood
- Myoglobin - oxygen storage in muslce
6
Q
Important historical fact about hemoglobin and myoglobin?
A
–First proteins whose 3D structure was determined by X-ray crystallography
–Model for the fact that protein sequence could lead to disease (Sickle cell anemia)
7
Q
What is myoglobin?
A
- Single peptide chain
- Arranged with several alpha helices, turns, and loops
- Great for O2 STORAGE
8
Q
What is hemeglobin?
A
- Has 2 alpha subunits and 2 beta subunits
- Each resemble a single myoglobin protein
- Great for O2 TRANSPORT
9
Q
What’s an important thing to note about the heme group?
A
- Not part of the amino acid
- Bound to the protein
- Hangs there via noncovalent bonds
10
Q
How many heme molecules per chain?
A
1
11
Q
What’s important to note about the alpha and beta subunits?
A
- They are a lot alike and similar to myoglobin
- They have NOTHING to do with alpha helices or beta sheets
12
Q
What is hemoglobin comprised of?
A
4 polypeptide chains
13
Q
How does the heme group bind to iron?
A
Each subunit has a heme cofactor that has a central Fe2+ that binds to O2
14
Q
How does oxygen binding work?
A
The central Fe2+ has 6 coordination sites
–> 4 to protoporphyrin
–>1 to His
–>1 to O2
15
Q
What is the difference between deoxyhemoglobin and oxyhemoglobin?
A
- Deoxy = “without oxygen”
16
Q
Where is iron located in deoxy-hgb vs oxy-hbg?
A
- In deoxyhemoglobin, the Fe2+ is outside the plane of the heme
- When the iron binds to oxygen, it shifts into the heme plane
17
Q
How does oxygen binding affect everything in the hemoglobin?
A
- Allows Fe into heme plane (pulls Fe up and into the plane)
- Tugs on His attached to it
- His is one aa in the hgb protein so this in turn tugs an alpha helix peptide chain
18
Q
What happens on the other side of the heme when oxygen is bound?
A
The bound oxygen is stabilized by H-bonding to another His
19
Q
What’s an important thing to note about histidine?
A
Histidine side chains are part of each subunit and part of the complete protein
20
Q
What is the relevance of functional magnetic resonance imaging (fMRI)?
A
Detects increased neuronal activity (local) is accompanied by increase oxyhemoglobin perfusion
–Important because stimulus induced brain activity can be measured
21
Q
What else is changed by the presence of oxygen?
A
- Structural changes
- magnetic properties of hemoglobin
–> changes in the ratio of the paramagnetic state to the diamagnetic state can be detected
–> essentially when brain is functioning/thinking, change in oxygen scan state
22
Q
How well does myoglobin bind to oxygen?
A
Very well (high affinity) even in low levels
–O2 levels must be very low before O2 comes back off
23
Q
What does the myoglobin O2 binding curve look like?
A
Hyperbolic
24
Q
What does the curve of hemoglobin suggest compared to myoglobin?
A
- Sigmoidal binding curve of hemoglobin reveals more dynamic binding of O2 compared to myglobin
- Suggest different binding mechanism (cooperative binding)
25
What is cooperative binding?
Hemoglobin is comprised of 4 subunits -- as it binds to oxygen, it increased ability for next oxygen to bind to hemoglobin
--does this by changing shape every time an oxygen is bound
26
Does deoxy-hemoglobin cooperatively bind and does it do it well?
Yes it cooperatively binds. It's poor in o2 bind but binding gets easier as more oxygen is attached
27
Can cooperative binding work in reverse?
Yes. Once fulled loaded hemoglobin lets go of one oxygen, it lets go of the next more easily
28
How are the subunits affected by cooperative binding?
Change shape upon O2 binding
--there is a 15 degree rotation of one pair of dimers (alpha1beta1) relative to the other pair (alpha2beta2)
29
How does O2 affect conformation of the protein
Pulls Fe, which pulls His, which pulls peptide chain. Subtle change in confromation favors O2 binding of over subunits
30
What are T and R states of proteins in general terms
1. T and R are two different physical "conformations" of the protein subunit
2. T and R designation used generally to discuss "states" of many proteins
31
What is the difference between T and R states of a hemoglobin?
1. T (tense state) has lower affinity, less likely to bind oxygen (see with Deoxy-hgb)
2. R (relaxed state) more active, higher affinity and more likely to bind oxygen (see with Oxy-hg)
32
In what locations is hemoglobin most in T state and R-state
1. In T state at bottom of graph, near origin
2. In R state near top of graph
33
How does O2 binding different depend on location?
1. Hemoglobin binds oxygen best when lots of oxygen is present (in lungs)
2. Binds poorest when oxygen is lower (peripheral tissues)
34
What's a more in depth description of the Bohr affect
1. Tissues that work harder reserve more oxygen
(enhanced oxygen delivery to tissues as metabolism increases)
--effects are due to H+ and carbon dioxide (promote the T state of hemoglobin)
35
What type of effectors are H+ and CO2
Allosteric effectors -- bind to hemoglobin in places other than oxygen binding sites to regulate the oxygen carrying capacity of hemoglobin
--changes shape, causing more oxygen to be released
36
Empty
Empty
37
How does CO2 promote T state?
1. Reacts with terminal amino groups of the proteins near the interface between alpha beta dimers
2. Stabilizes deoxyhemoglobin
--Induces a change in charge from positive (or neutral) to a negative charge, causing a change in confirmation to one that favors T state
38
How exactly do H+ and CO2 encourage O2 release?
1. Stabilizing the T-state
--This shifts the sigmoidal curve
39
Compared to hemoglobin, what are subunits and binding behavior like in myoglobin?
1. Only one subunit
2. No cooperative or allosteric behavior
3. In muscle cells
40
How does myoglobin bind the O2 in muscle cells?
Binds the O2 that hemoglobin releases as blood goes by the muscle cells (its affinity for O2 is higher)
41
Can myoglobin deliver O2?
No, but it DOES store O2 better compared to hemoglobin
42
What's the process for sickle cell to "form"?
Deoxygenated (T-state) hemoglobin S of sickle cell anemia creates a hydrophobic patch on the protein surface
--this hydrophobic patch isn't on surface in R state
--mutation affects a surface aa on the beta chain
--Glu(-) is changed to val at amino acid 6 (hydrophobic)
43
What are the effects of sickle cell anemia?
1. Hemoglobin becomes fibrous
--mutation alters solubility (primarily in T-state) and promotes aggregation between hemoglobin proteins which assemble into strands and fibers (most dramatic in deoxy form)
44
What happens when fibers form?
They reshape RBS into spiny, sickle shaped cells: damage the RBC membrane, cells lodge in blood vessels, clots form, organ damage may result, extreme pain, becomes medical crisis
45
What are enzymes?
1. Proteins
2. Also called biological catalysts
3. Not consumed or altered in a reaction
46
What's the basic formula for an enzymatic reaction?
E + S ---> ES ---> P
47
Where do substrates bind?
Bind on a specific location on enzyme known as "active site"
48
What is an active site
1. A 3D cleft formed from different parts of the amino acid sequence
2. Takes up a small part of the total volume of enzyme
49
What environment is an active site typically found in?
1. Nonpolar -- water is excluded (unless its a substrate, i.e proteases)
50
What are the two important features of enzymes?
1. Catalytic power
2. Specificity for activity of enzyme
51
What is catalytic power?
Enzymes increase rate of reactions (decreasing time it takes to reach equilibrium)
52
How many steps do enzymes go through and why?
Enzymes are not consumed/altered in reaction. so they cycle through 3 steps
1. Bind substrate
2. Chemical change
3. Release
53
What is an example of enzyme specificity?
Proteases -- enzymes that hydrolyze proteins into smaller peptides or amino acids
54
What are the 3 types of proteases and their specificity?
1. Chymotrypsin: aromatics, L, M
2. Trypsin: K, R
3. Papain: any peptide bond
55
How is specificity dictated?
Dictated by the specific enzyme subtrate interactions
56
What is gives chymotrypsin its specificity?
Hydrophobic pocket interaction with phe
57
What is a catalytic triad?
It's responsible for cutting the targeted peptide bond (catalytic power)
--Many proteases cut peptide bonds --> catalytic triad is a common structure in the active site of these enzymes
58
How is the specificity of an enzyme made?
Specificity of enzyme is gained through the precise interaction of the enzyme with the amino acid R group of substrate
59
How is catalytic power achieved?
Catalytic power is achieved through interaction of peptide bond of substrate with the catalytic triad (Ser, His, and Asp)
60
What's the importance of interactions between peptide bond of substrate with catalytic triad?
1. Correct positioning of substrate
2. Energenic stabilization
3. Lowering of free energy of activation
61
What are cofactors and why are they important?
1. Catalytic activity of many enzymes require a cofactor
62
What are the two types of cofactors
1. Coenzymes : organic, non-protein, most are derived from vitamins
2. Metals : metal ions, trace minerals used by enzymes for catalysis
63
What are two thermodynamic properties of a reaction?
1. Free energy difference (delta G) between products and reactants
2. Energy required to initiate the conversion of reactions into products (activation energy)
64
What does delta G tell us?
If the reaction will occur spontaneously
--measure of the amount of mechanical or chemical work that can be extracted from a system, chemical compound, reaction, or series of reactions
65
What does activation energy tell us?
Determines the rate of a reaction
66
Can enzymes affect delta G or activation energy?
Enzymes can ONLY affect activation energy
67
What is the second law of thermodynamics?
1. Total entropy of a system and its surroundings is always increasing
Delta G = Delta H - T(DeltaS)
2. Used to describe energetics of systems and even biological reactions
68
When will a process or reaction proceed spontaneously?
If change in free energy is negative (releases energy)
--Negative delta G = exergonic
--On graph substrate is above product
69
What does it mean if delta G = 0?
Reaction is at equilibrium (neither produces or consumes energy)
--It is NOT static
70
What does it mean if delta G is positive?
1. It's endergonic (takes energy)
2. Reaction will not go forward on its own; energy must be put into the system to force the reaction
3. Reaction is non-spontaneous
71
What are coupled reactions?
When reactions have positive G, they couple endergonic reaction to exergonic reactions so that the overall delta G is negative
72
What is an example of coupled reactions?
DNA polymerase activity is coupled with PPi hydrolysis by pyrophosphatase to make the overall reaction spontaneous
**Living systems couple endergonic (polymerization of DNA) to exergonic spontaneous reactions (hydrolysis of PPi)
73
If you know delta G of the forward reaction, do you know delta G of the reverse?
Yes.
A --> B ; G = x
A <-- B ; G = -x
74
What does amount of activation energy have NO effect on?
1. Free energy of the substrate
2. Free energy of the product
3. Delta G of the overall reaction
75
How do enzymes lower the activation energy?
Enzymes work by stabilizing the transition state, thus lowering the activation energy
--reduce energy required to reach transition state
76
Does an enzyme alter the equilibrium of a reaction?
No -- just affects whether the reaction will reach equilibrium sooner or slower
77
What is an ES complex?
During an enzymatic reaction, and ES complex is formed
--Depicted in 3 ways
1. E + S --> <-- ES --> E + P
2. The 3 steps of a cycle
3. Activation energy
78
What is a common feature of active sites?
Substrates are bound to enzymes by multiple weak interactions
**H bonds directionality reinforces specificity
79
What's an important note about the noncovalent interactions of an enzyme?
The noncovalent interactions between "substrate" and enzyme IN the TRANSITION state are STRONGER than the interactions of enzyme and substrate in the GROUND state
80
What does specificity for the substrate depend on?
Exact arrangement of atoms lining the active site
81
What are the two models of enzyme substrate binding?
1. Emil Fischer's Lock and Key Model
2. Dan Koshland's Induced Fit Model
82
What is the difference between Lock and Key Model and Induced Fit?
1. Lock and Key: alignment between S and E generate multiple non covalent interactions
2. Induced Fit: Van der Waals become significant when many atoms of substrate and enzyme come close together -- so shapes are complementary
**for induced: binding of substrate causes transient change to shape of enzyme, reinforcing weak interactions
83
What does the induced fit model do to substrate and enzyme
Increases noncovalent interactions between S and E
84
How is an E-S complex formed?
1. Aligning the substrate with the enzyme so the enzymatic reaction can happen
2. Desolvation -- stripping off water molecules from the groups that need to come close together (water decreases many non covalent interactions)
3. Induce strain in bonds that need to break
**ALL of these processes have positive delta G
85
Given that the formation of an E-S complex includes positive and unfavorable processes, where does the energy come from?
Lowering of activation energy (compared to teh same reaction in the absence of enzyme) comes from additive effect of many weak, reversible non-covalent interactions between substrate and enzyme in the transition state
86
What is delta G knot'
Delta G under standard conditions
--pH = 7, 1 M, 1 atm, 25 degrees celsius)
87
What is an isomerization?
Rearrangement of atoms to form isomers
88
If the reaction won't proceed/isn't spontaneous, will the presence of an enzyme change that?
No. Enzymes cannot defy laws of thermodynamics
89
What is the mathematical equation related to delta G?
G = G' + 1.37log10([product]/[substrate])
90
What's the difference between Keq and Q?
1. Keq = equilibrium constant quotient at standard conditions
2. Q = mass action quotient at NON standard conditions
91
What happens as [S] increases?
The rate of the reaction increases until a maximum rate is reached and the enzyme is saturated
92
What is Vmax?
The maximum rate the enzyme can attain
**total number of all enzyme products made
93
What is Km?
The Michaelis constant
94
What is a Lineweaver-Burk Plot?
A more accurate way of determining Vmax and Km
**These are double reciprocal plots and are especially useful when looking at inhibition of enzymatic reactions
95
What is the difference between Michael Menton and Lineweaver Burk graphs?
1. Michaelis Menton is a log graph
2. Lineweaver Burk is a linear -- y intercept = 1/vmax, x intercept = -1/km
96
What does Km mean?
1. Km is the [S] at 1/2 Vmax
2. Essentially describes strength of E and S binding
97
What does a small vs large Km value indicate?
1. Small Km value indicates that enzyme requires only a small amount of substrate to reach max velocity (greater/tighter binding affinity of E for S)
2. Large Km indicates need for high substrate concentrations to achieve maximum reaction velocity (weak binding)
98
What can you compare Km to?
1. Useful to compare Km for different substrates for one enzyme
2. Useful to compare Km for a common substrate used by several enzymes
99
What is the turnover number
1. Measure of catalytic activity and a different way of expressing Vmax (use Kcat instead)
2. Essentially measures number of reactions ONE enzyme does (per second) when enzyme is saturated with substrate
100
What are allosteric enzymes?
Class of enzymes whose catalytic activity is not constant or is regulated
--typically have multiple active sites --> binding multiple S and/or activators or inhibitors
101
What happens during allosteric enzyme process?
Following binding with a regulatory substance, there is a change in the shape of the enzyme and a corresponding increase or decrease in activity
102
Do allosteric enzymes follow M-M kinetics?
NO --> Michaelis Menton kinetics cannot describe all enzymes
103
Why do allosteric enzymes have multiple binding sites and what is a direct result of that?
1. Because they have multiple subunits
2. Binding of one S enhances binding affinity of 2nd S
104
What do the active sites display for allosteric enzymes?
Cooperativity, which shows in the sigmoidal V initial vs [S] curve
--often they have binding sites for activators or inhibitors, which shift the binding curve left or right respectively
105
Are the allosteric site and active site the same?
No. Allosteric site is found separate to the active site.
--active site binds substrate and converts to product
--allosteric binds regulator which results in shape change and thus enzyme speed
106
What is the difference between an inhibitor and an activator?
1. Activator : speeds up reaction
2. Inhibitor : slows down reaction
107
What do enzymes do in a chemical reaction
Permit, acceleration, and sometimes control
**should note that enzymes themselves are controlled (important for spontaneous reactions)
108
What is enzyme inhibition
Decrease enzyme's ability to bind substrate, decrease catalytic activity (turnover) or both
109
What are examples of enzyme inhibition and why is it important?
Most drugs, (Tylenol, Ampicillin, etc), toxins, venoms and chemical warfare agents
--Kinetics studies are used to study effectiveness or potency
110
What type of mushroom is an example of enzyme inhibition and what does it do?
1. Amanita mushroom: contains potent toxin alpha amanitin
2. Alpha amanitin inhibits translocation activity of RNA polymerase (essentially stops ability to make protein that you need)
111
What are the two types of enzyme inhibition?
1. Reversible : molecule binds to an enzyme to inhibit it, but can be released
**Inhibitor binds and dissociates from enzyme in an equilibrium process
2. Irreversible : a molecule that causes inhibition that cannot be reversed or reverses very slowly
112
What are the three descriptives of reversible inhibitors?
1. Competitive
2. Non-competitive
3. Uncompetitive
113
What is a competitive inhibitor?
1. Resembles S structurally but is not a substrate and competes with substrate for the active site
114
How do competitive inhibitors affect the enzyme and can these effects be overcome?
1. binding of CI reduces # of enzyme molecules that can bind S
2. Effects can be overcome by high [S] since both compete for binding in the active site
115
How do competitive inhibitors affect Km and Vmax?
1. Km is increased
2. Vmax can be attained (remains constant)
116
What is a non competitive inhibitor?
Do not resemble S, bind at a different site on E and decrease its catalytic activity
**doesn't depend on order of binding
117
What are some effects of non competitive inhibitors and can it be overcome?
1. Substrate can still bind to enzyme inhibitor forming enzyme inhibitor substrate but cannot form product
2. NCI can NOT be overcome by high [S]
118
How does non competitive inhibitors affect Km and Vmax
1. Km remains constant
2. Vmax can NOT be attained
119
What is an uncompetitive inhibitor?
Bind only to enzyme substrate complex (NOT in the active site)
120
What are the effects of uncompetitive inhibitors and can it be overcome?
1. Enzyme substrate form then enzyme substrate inhibitor forms and cannot form product
2. UCI can NOT be overcome by high [S]
121
How does un competitive inhibition affect Km and Vmax?
1. Lowers Km
2. Lowers Vmax
122
Which type of graph is used to visualize and quantitatively compare the kinetics of reversible inhibition?
Lineweaver Burk
123
What is the difference between a competitive inhibitor plot, noncompetitive inhibitor plot, and uncompetitive plot?
1. Competitive inhibitor : two lines intersect at y axis
2. Non-competitive inhibitor: two lines start at same Km (intersect at x axis?)
3. Uncompetitive: lines remain parallel to one another
124
What is an irreversible inhibitor?
Bind an enzyme to inactivate it, and do not release from the enzyme
125
What is an example of an irreversible inhibitor?
Suicide inhibitors
126
How do suicide inhibitors work?
Bind the active site and a reaction is catalyzed to produce the actual inhibiting compound -- catalytic product is the actual inhibitor
--many times these reaction with R groups of the enzyme
OSU
flashcards
Decks in class (24)
# Cards
-
IDS30
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IDS 230
-
Ch 1-3 Microbio70
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Ch 4-5 Microbio49
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Lab Practical 1 Prep71
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Micro Exam 2109
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Lecture 694
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Lab Practical 2117
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Exam 3 Microbio123
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Animal Bio exam 346
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Exam 4 Micro85
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Bio Practical 395
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Bio Final1
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Micro final91
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Micro Final Sec 2120
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Micro Final Sec 473
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Micro Final Sec 648
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Biochem Exam 1105
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Biochem 2126
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Biochem 3166
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Biochem 4177
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Glycolysis1
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Pathways1
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Biochem 418
