biochem exam 1 Flashcards
(200 cards)
1
Q
At high S, velocity does not depend on?
A
S
2
Q
How do you determine kinetic parameters Km and Vmax?
A
Nonlinear Michael’s-Menten plot
3
Q
What is good for analysis of two-substrate data or inhibition?
A
Lineweaver-Burk plot
4
Q
What is a Lineweaver-Burk plot?
A
Linearized double-reciprocal plot
5
Q
How do we get a Lineweaver-Burk plot?
A
Taking the reciprocal of both sides of the Michaelis-Menten equation
6
Q
What is maximum velocity (Vmax)?
A
The highest speed the enzyme can work
7
Q
What is the Michaelis constant (Km)?
A
The point at which the enzyme is working at half their maximum speed
Each enzyme is unique in its ability to handle workloads, like people!
8
Q
What does a low Km say about the enzyme?
A
It’s really fast
9
Q
What 3 things does the Michaelis-Menten equation tell us?
A
- Reaction rate (v) is based on substrate concentration [S].
- If there’s only one enzyme, they have a limit (Vmax) no matter how many substrates show up.
- Km tells us how quickly the enzyme reaches half of their full speed.
10
Q
Why do we like the Lineweaver-Burk plot?
A
The Lineweaver-Burk plot is a shortcut to straighten the curve into a line, making it easier to read important values.
11
Q
What are we being told where the line crosses the y-axis (1/Vmax) on the Lineweaver-Burk plot?
A
Tells us the enzyme’s fastest possible speed.
12
Q
What are we being told where the line crosses the x-axis (-1/Km) on the Lineweaver-Burk plot?
A
Tells us how much substrate is needed to get halfway to full speed.
13
Q
What are we being told where the slope of the line (Km/Vmax) on the Lineweaver-Burk plot?
A
Tells us how efficient the enzyme is.
14
Q
Michaelis-Menten vs Lineweaver-Burk: which one is good for visualizing real-life enzyme behavior?
A
Michaelis-Menten
15
Q
Michaelis-Menten vs Lineweaver-Burk: which one is good for making precise calculations?
A
Lineweaver-Burk
16
Q
What is the ideal reaction rate?
A
Maximum theoretical rate at which an enzyme can work under perfect conditions
17
Q
Where do we see ideal reaction rate in a Michaelis-Menten curve?
A
This is the flat part at the top where the enzyme is going as fast as it possibly can.
18
Q
What are the 4 assumptions we make?
A
- Steady state assumption
- Simple enzyme kinetics
- Excess substrate available
- Initial velocity conditions
19
Q
What do we mean by steady state assumption?
A
The amount of enzyme bound to substrate (ES complex) stays constant because it’s being formed and broken down at the same rate
20
Q
What describes how many substrate molecules can one enzyme molecule convert per second?
A
kcat (turnover number)
21
Q
What describes an approximate measure of substrate’s affinity for enzyme?
A
Km (Michaelis constant)
22
Q
What are compounds that decrease enzyme’s activity?
A
Inhibitors
23
Q
What are irreversible inhibitors?
A
Inactivators that react with the enzyme
24
Q
What are reversible inhibitors?
A
Inhibitors that bind to and can dissociate from the enzyme
25
What kind of inhibitor often acts as powerful toxins?
Irreversible inhibitors, because one inhibitor molecule can permanently shut off one enzyme molecule
26
What kind of inhibitor is often structural analogs of substrates or products?
Reversible inhibitors
27
Reversible inhibitors can bind to ____________ to prevent the binding of the substrate or _____________ to prevent the reaction.
The free enzyme; the enzyme-substrate complex
28
What are competitive inhibitors?
Inhibitors that compete with the substrate for binding
29
True/False: Competitive inhibitors do NOT affect catalysis.
TRUE
## Footnote
Competitive inhibitors only block substrate binding by competing for the active site, but they don't interfere with the actual reaction mechanism once the substrate successfully binds.
30
What does it mean for the enzyme that competitive inhibitors do not affect catalysis?
This means that if enough substrate is added, the enzyme can still work at full efficiency because the inhibitor gets 'outcompeted' and pushed aside.
31
How does competitive inhibition impact Vmax and Km?
No change in Vmax; apparent increase in Km
32
What do competitive inhibitors do to the Lineweaver-Burk plot?
Lines intersect at the y-axis
33
What do competitive inhibitors do?
Work by blocking the active site
34
What do noncompetitive inhibitors do?
Act by attaching to an allosteric site, changing the shape of the enzyme so it can no longer bind to the substrate
35
What are noncompetitive inhibitors also called?
Allosteric inhibitors
36
What does mixed inhibition bind to?
Binds to the enzyme with or without the substrate
37
What does mixed inhibition bind to and inhibit?
Regulatory site; inhibits both substrate binding AND catalysis
38
What does mixed inhibition do to the Lineweaver-Burk plot?
Lines intersect left from the Y-axis
39
What does mixed inhibition do to Vmax and Km?
Decreases Vmax, apparent change in Km
40
What is a mixed inhibitor that does NOT change Km?
Noncompetitive inhibitors
41
What undergoes induced fit on substrate binding?
Hexokinase
42
Hexokinase starts in a ______ shape, then __________________________ when it binds to _______________.
U; the ends pinch towards each other; D-glucose
43
How does hexokinase demonstrate the role induced fit plays on reaction rate?
Since hexokinase changes shape upon binding, the rate of reaction isn't just about simple binding—it also involves this conformational shift, which affects the overall reaction speed.
44
What are charged intermediates stabilized by?
Transfer protons to or from the substrate or intermediate to form a species that breaks down more readily to products
45
How does His work in acid/base catalysis?
MVP because it can both donate and accept protons depending on the pH.
46
How does Glu, Asp work in acid/base catalysis?
Negatively charged, great at pulling protons (acting as a base).
47
How does Lys, Arg work in acid/base catalysis?
Positively charged, great at donating protons (acting as an acid).
48
How does Cys work in acid/base catalysis?
Has a -SH (thiol) group that can donate or accept protons like a champ.
49
How does Ser work in acid/base catalysis?
Have -OH (hydroxyl) groups that sometimes help in proton transfers.
50
How does Tyr work in acid/base catalysis?
Can sometimes donate protons using its phenol (-OH) group.
51
Give an example of glutamate and aspartate in action breaking down bacterial cell walls.
Lysozyme (Breaks Down Bacteria)
Glutamate (Glu 35) donates a proton → acts as an acid.
Aspartate (Asp 52) stabilizes the reaction → acts as a base.
Result? It chops up bacterial cell walls like a boss.
52
What is covalent catalysis?
A transient covalent bond between the enzyme and the substrate
53
What does covalent catalysis involve?
Temporarily forming a covalent bond to stabilize the reaction.
54
What does covalent catalysis require?
A nucleophile on the enzyme
55
What is a nucleophile?
A chemical species that donates an electron pair to an electrophile to form a chemical bond in relation to a reaction
56
Which amino acid side chains can act as nucleophiles in covalent catalysis?
Can be a reactive serine, thiolate, amine, or carboxylate
57
What is the cleavage of peptidoglycan by lysozyme?
A successive Sn2 steps model
58
In the first step of cleavage of peptidoglycan, what acts as a nucleophile?
Asp 52
59
What does Asp 52 attack in the first SN2 step?
Anomeric carbon (a carbon double-bonded to an oxygen)
60
After Asp 52 attacks the anomeric carbon, what does Glu 35 do?
Acts as a general acid and protonates the leaving group in the transition state
61
What happens after Glu 35 protonates the leaving group in the transition state?
Water hydrolyzes the covalent glycosyl-enzyme intermediate
62
Once water hydrolyzes the glycosyl-enzyme intermediate, what does Glu 35 do?
Acts as a general base to deprotonate water in the second SN2 step
63
What does it mean that some enzymes use multiple types of regulation?
Most enzymes aren't just 'on' or 'off'—they are carefully controlled using different regulatory mechanisms.
64
What is an example of insulin's allosteric regulation?
Phosphofructokinase-1 (PFK-1) is indirectly activated by insulin because insulin increases levels of fructose-2,6-bisphosphate, which is an allosteric activator.
65
What is an example of insulin's covalent regulation?
Insulin triggers dephosphorylation of key metabolic enzymes, like how it inhibits glycogen phosphorylase (which breaks down glycogen) by dephosphorylation.
66
67
What can interaction strength be expressed as?
Association and dissociation constants.
68
What is the association constant?
Quantifies the affinity of the ligand for the protein. A higher Ka means stronger binding.
69
The association (binding) constant is ____, units ____
Ka; M-1
70
The dissociation constant is _______, units _____
Kd; M
71
Give the formula for Kd's relationship to Ka.
Kd = 1/Ka
72
The formula for the association constant (Ka) describes the equilibrium between a ___________ and a _____________ forming a ______________.
Protein (P); ligand (L); protein-ligand complex (PL)
73
Give the formula for the association constant.
Ka = [PL]/[P][L]
## Footnote
[PL] = Concentration of the protein-ligand complex.
[P] = Concentration of free (unbound) protein.
[L] = Concentration of free (unbound) ligand.
74
The association constant formula follows the law of mass action, which states that _________________________________
At equilibrium, the ratio of the concentrations of products to reactants is constant.
75
Ka reflects what about the protein and ligand?
How much of the protein and ligand exist in the bound state relative to their free forms.
76
A HIGHER Ka means _________________________________
More of the protein and ligand are in the bound form (PL), indicating a stronger affinity between the two.
77
A LOWER Ka suggests _______________________________
Weaker binding, meaning that most of the protein and ligand remain unbound.
78
The dissociation constant Kd is the ___________ of Ka.
Inverse.
79
Give the formula for the dissociation constant.
Kd = [P][L]/[PL]
80
What is the Kd value for strong binding?
Kd < 10 nM
81
What is the Kd value for weak binding?
Kd > 10 mM
82
Protein: Avidin (egg white), ligand: _____________, Kd (M): _____________
Biotin; 1x10^-15
83
Protein: insulin receptor (human), ligand: _____________, Kd (M): _____________
Insulin; 1x10^-10
84
Protein: anti-HIV immunoglobulin (human), ligand: _____________, Kd (M): _____________
Gp41 (HIV-1 surface protein); 4x10^-10
85
Protein: nickel-binding protein (E. coli), ligand: _____________, Kd (M): _____________
Ni2+; 1x10^-7
86
Protein: calmodulin (rat), ligand: _____________, Kd (M): _____________
Ca2+; 3x10^-6
87
Specificity in protein binding refers to _____________________________
A protein's ability to selectively bind to a particular ligand or molecule among many possible candidates.
88
What does a measure of specificity tell us?
It describes how well a protein can distinguish its preferred ligand from other similar molecules.
89
What's the difference between affinity (Ka or Kd) and specificity?
Affinity (Ka or Kd) measures how tightly a protein binds to a ligand. Specificity measures how selectively a protein binds to its correct ligand over others.
90
Proteins recognize specific ligands based on 4 things:
1. Shape 2. Complementarity 3. Charge interactions 4. Hydrophobic/hydrophilic interactions.
91
What are the 2 models for specificity?
1. Lock and key 2. Induced fit
92
Who came up with the lock and key model?
Emil Fisher
93
What does the lock and key model assume?
The protein has a rigid binding site that only fits one ligand.
94
Who came up with the induced fit model?
Daniel Koshland
95
What does the induced fit model assume?
The protein can undergo conformational changes to better accommodate a specific ligand.
96
What is the adaptation called where conformational changes may occur upon ligand binding?
Induced fit.
97
What are the 2 things that induced fit allows for?
1. Tighter binding of the ligand 2. High affinity for different ligands.
98
True/false: In the induced fit model, both the ligand and the protein can change their conformations.
True!
99
When it comes to specificity (regardless of the model), what can we generally assume about discrimination between ligands?
A highly specific protein will bind strongly to its intended ligand and weakly (or not at all) to structurally similar molecules.
100
_______________ are a family of oxygen-binding proteins.
Globins.
101
Knowing what we know now about affinity and specificity, why is CO (carbon monoxide) so toxic?
CO coordinates to heme iron with greater affinity than does O2. When a molecule of CO is bound to heme, O2 is excluded.
102
Why does His F8 bind to Fe2+ in hemoglobin?
To keep it from oxidizing to Fe3+ (oxidation is gaining an electron).
103
What kind of bond do we see between His and Fe2+ in hemoglobin?
Covalent.
104
Explain what would happen without HisF8.
Without His F8, Fe²⁺ would be too unstable and could easily be oxidized, permanently trapping O₂ as Fe³⁺-O₂⁻ (methemoglobin).
105
How does the distal His (His E7) bind to Oxygen?
Hydrogen bonding.
106
Why does His E7 have to bind to O2 to facilitate O2 binding to Fe2+? (4 reasons)
1. Prevents Fe²⁺ oxidation by stabilizing the O₂ molecule 2. Lowers CO binding affinity, reducing poisoning risk 3. Optimizes O₂ binding affinity for effective transport 4. Allows reversible binding, ensuring O₂ delivery to tissues.
107
Hemoglobin and myoglobin are both ________________ but hemoglobin is a _______________ whereas myoglobin is a _____________________
Globular proteins; tetramer; polypeptide chain.
108
What does it mean that hemoglobin is an a2b2 tetramer?
It has 2 identical alpha and beta chains.
109
A heme group is made of 2 things:
A protoporphyrin ring and a central iron atom.
110
What is allosteric regulation?
Allosteric regulation refers to the process where the binding of a molecule at one site on a protein affects the activity at another site on the same protein.
111
What is it called when a molecule binds somewhere other than the active site and affects function?
Allosteric regulation.
112
Allosteric regulation can be ____________ or ______________.
Positive or negative.
113
Positive allosteric regulation means the binding of a molecule at one site on a protein will ________________ the activity at another site, whereas negative will _______________________ activity.
Activate; inhibit.
114
True/false: Cooperativity is a type of allosteric regulation, but not all allosteric regulation involves cooperativity.
True!
115
What are the 2 types of allosteric proteins?
1. Homotropic 2. Heterotropic.
116
Homotropic means:
Normal ligand of the protein is the allosteric regulator.
117
Heterotropic means:
Different ligand affects binding of the normal ligand.
118
What is cooperativity?
Cooperativity is a specific type of allosteric regulation when one ligand binding affects binding at other sites that occurs in multi-subunit proteins (like hemoglobin).
119
Positive cooperativity means _________________________
Ligand binding increases affinity for additional ligands (e.g., hemoglobin and O₂).
120
Negative cooperativity means _________________________
Ligand binding decreases affinity for additional ligands.
121
What does it mean that hemoglobin binds oxygen cooperatively? (positive cooperativity)
First binding makes it easier for more to bind, so once one O₂ binds, it gets easier for the next ones.
122
Describe negative cooperativity.
First binding makes it harder for more to bind.
123
Describe an example of negative cooperativity.
Glucokinase - Once a molecule binds, the shape change makes additional binding less favorable.
124
Describe non-cooperative binding.
Binding at one site doesn't affect other sites.
125
Give an example of non-cooperative binding.
Myoglobin & O₂ - Each O₂ binds independently with no change in affinity.
126
Is this homo or heterotropic allosteric regulation: A separate molecule acts as an activator or inhibitor.
Heterotropic.
127
Is this homo or heterotropic allosteric regulation: The ligand affects itself.
Homotropic.
128
Is this homo or heterotropic allosteric regulation: The same molecule that binds is also the one regulating activity.
Homotropic.
129
Is this homo or heterotropic allosteric regulation: CO₂ & H⁺ in hemoglobin (Bohr Effect) - They bind away from the O₂ site and cause O₂ release.
Heterotropic.
130
Hemoglobin (Hb) is a ____________ of two subunits (a2b2).
Tetramer.
131
Is this homo or heterotropic allosteric regulation: O₂ in hemoglobin - Once one O₂ binds, it helps (positive cooperativity).
Homotropic.
132
Is this homo or heterotropic allosteric regulation: A different molecule binds and changes function.
Heterotropic.
133
Each subunit of hemoglobin is similar to ______________.
Myoglobin.
134
True/false: Hemoglobin and myoglobin have very similar sequences.
False! Only some amino acids are conserved in all known globins, and these conserved residues likely play a critical role in oxygen binding, heme stabilization, or protein folding.
135
What do proteins with common functions share even across different species?
Proteins with a common function (O₂ binding) share conserved residues, even across different species.
136
When it comes to proteins with common functions, what is more important than exact sequence similarity?
Structural similarity is more critical than exact sequence similarity.
137
What do we need to remember about key functional residues when it comes to proteins with common functions?
Key functional residues (e.g., distal His E7) stay in the same structural position even if their sequence number differs.
138
The common helix-letter-and-number designation for amino acids in proteins with common functions does not necessarily correspond to _________________________________________________________
A common position in the linear sequence of amino acids in the polypeptides.
139
Hemoglobin has 2 conformations: ___________ and _____________
T and R.
140
T means _______ and R means _____________
Tense; Relaxed.
141
When in the T state, hemoglobin has a __________ affinity for O2, and when it's in the R state, it has a _______________ affinity for O2.
Lower; higher.
142
What conformational change is triggered by O2 binding? What does this involve?
T to R; involves breaking ion pairs between the α1-b2 interface.
143
Actively metabolizing tissues generate ____, __________ the pH of the blood near the tissues relative to the lungs.
H+; lowering.
144
Hb Affinity for oxygen depends on ____________.
The pH.
145
H+ binds to Hb and stabilizes the _________.
T state.
146
When H+ binds to Hb, this protonates ______ which then forms a __________ with _________
His146; salt bridge; Asp94.
147
When a salt bridge is formed between His146 and Asp94, this leads to _______________.
The release of O2 (in the tissues).
148
The pH difference between _________ and _________________ increases efficiency of the O2 transport.
Lungs and metabolic tissues.
149
______________ describes how changes in pH and CO₂ levels affect hemoglobin's oxygen-binding affinity.
The Bohr effect.
150
The pH of blood is _____ in the lungs and _____ in the tissues. Experimental measurements on hemoglobin binding are often performed at pH ______.
7.6; 7.2; 7.4.
151
_______________ is a molecule that binds to hemoglobin and makes it release oxygen more easily.
2,3-Bisphosphoglycerate.
152
What kind of regulator is 2,3-Bisphosphoglycerate?
Negative heterotropic regulator of Hb function.
153
How is 2,3-Bisphosphoglycerate produced?
From an intermediate in glycolysis.
154
How does 2,3-Bisphosphoglycerate work?
Small negatively charged molecule, binds to the positively charged central cavity of Hb and stabilizes the T state.
155
what does heterotrophic mean?
different ligand affects binding of the normal ligand
156
2-3 BPG is found is RBC's (erythrocytes) at very ______ concentrations, typically around _______
high; 5 mM
157
specifically, BPG binding stabilizes the T state of _________
deoxyhemoglobin
158
hemoglobin binds to oxygen quite tightly when BPG is __________
entirely absent
159
at high altitudes, O2 delivery _________
declines
160
how much does O2 delivery decline at high altitudes?
by about 1/4th, to 30% of maximum
161
increase in BPG concentrations _________ the affinity of hemoglobin for O2, so approximately _____ is delivered to the tissues
decreases; 37%
162
what mutation in Hb causes sickle cell anemia?
Glu6 changes to Val in the b chain of Hb
163
what about the Hb mutation sickles the red blood cells
the new valine side chain can bind to a different Hb molecule to form a strand
164
what type of sickle cell anemia usually ends in death
untreated homozygous individuals generally die in childhood
165
what type of sickle cell anemia makes a person resistant to malaria?
heterozygous individuals exhibit a resistance to malaria
166
what is anemia?
a condition in which you dont have enough healthy RBC's to carry adequate oxygen to your tissues
167
describe how Hb strands form in sickle cell anemia
deoxyhemoglobin S has a hydrophobic patch on its surface, which causes the molecules to aggregate into strands that align into insoluble fibers
168
what is the difference between hemoglobin A and hemoglobin S?
a single amino acid change in the beta chains
169
what causes deoxyhemoglobin S to aggregate into strands?
a hydrophobic patch on its surface due to the amino acid change
170
what do the aggregates of deoxyhemoglobin S form?
insoluble fibers
171
enzymes are _______
catalysis
172
what do enzymes do?
increase reaction rates without being used up
173
most enzymes are _______ proteins
globular
174
t or f: some RNA (ribozymes and ribosomal RNA) also catalyze reactions
True! this is the exception to the globular protein
175
study of enzymatic processes is the oldest field of biochemistry, dating back to ________
late 1700s
176
what is the protein part of an enzyme called?
apoenzyme
177
what do we call an active enzyme including coenzyme and metal ions?
holoenzyme
178
what do we call the key that turns the enzyme on?
cofactors/ coenzyme
179
whats an example of a cofactor for Luciferin?
Mg2+
180
whats an example of an enzyme for Luciferin?
Luciferase
181
what do we call a hydrogen anion
hydride
182
what class of enzyme often forms a new double bond or a new ring structure
Lyases
183
enzymes increase reaction rates (k) by
decreasing ΔG‡
184
what is ΔG‡?
activation barriers, slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction
185
do enzymes effect equilibrium?
no
186
what is ΔG?
equilibrium
187
what is the michealis constant?
the substrate concentration at which the reaction is at half its maximum velocity, VmaxV_{\max}Vmax
188
what is the michealis equation?
v=kcat[E][S]/Km+[S]
v= reaction velocity
Kcat=catalytic rate constant
E=concentration of the enzyme
S= concentration of the substrate
Km= michealis constant
189
the noncovalent enzyme substrate complex is known as the _______
Michaelis complex
190
how do enzymes act?
by binding substrates
191
how does the enzyme speed up reaction rate?
by lowering activation energy
192
name the enzyme class based on the reaction: Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor
ligases
193
name the enzyme class based on the reaction: transfer of groups within molecules to yield isometric forms
isomerases
194
name the enzyme class based on the reaction: cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds
lyases
195
name the enzyme class based on the reaction: group transfer reactions
transferases
196
what happens during a hydrolysis reaction?
transfer of functional groups to water
197
name the enzyme class based on the reaction: hydrolysis reactions
hydrolases
198
name the enzyme class based on the reaction: transfer of electrons
oxidoreductases
199
how do we define the classes of enzymes
by the reactions catalyzed
200
name the 6 classes of enzymes
oxidoreductases
transferases
hydrolases
lyases
isomerases
ligases
