Midterm 2 Flashcards
1
Q
What type of chaperone is GroEL–GroES?
A
chamber
2
Q
Many diseases, including Alzheimer’s disease, are associated with
A
protein aggregation
3
Q
Globular proteins are mostly ________ and fibrous proteins are mostly _________.
A
soluble
insoluble
4
Q
Which fibrous protein is a myosin coiled-coil protein?
A
keratin
5
Q
Describle keratin’s structure.
A
Left-handed coiled coil dimer
Right-handed helix
Disulfide bridges between 2 coiled coils
6
Q
Which fibrous protein is the aa sequence (G-A-S) repeated and tightly packed?
A
Silk
7
Q
Why does a Vitiman C deficiency cause scurvy?
A
Vitiman C is a cofactor for hyroxylase which hydroxylates proline forming HyPro. HyPro is a subunit of Collagen and the collagen deficiency causes scurvy.
8
Q
Which fibrous protein is (HyPro-Pro-Gly) repeated?
A
Collagen
9
Q
Protein folding is a ________ process.
A
spontaneous (exothermic)
10
Q
What are chaperone proteins and what 3 functions of chaperones?
A
Proteins that facilitate the formation of stable 3D sturctures.
- Help proteins fold properly
- Fix misfolded proteins
- Disrupt aggregates
11
Q
What are the 2 types of chaperone proteins?
A
- Clamp - heat shock
- Chamber -GroEL–GroES protein complex
12
Q
What are the 3 Models of Protein Folding?
A
- Hydrophobic collapse - clustering of hydrophobic side chains
- Framework model - 2º/3º structures form independently
- Nucleation model - localized interactions between 2º stuctures
13
Q
What are the 4 general steps involved in protein study?
A
- Cloning
- Expression
- Purification
- Characterization
14
Q
What is the specific activity of a protein?
A
Total amount of activity of the target protein divided by the total amount of protein in the fraction.
Specific activity INCREASES with fractionization
15
Q
As you centrifuge a cell, what are the 4 fractions you will get as you increase time and force (g)?
A
1st - Nuclear fraction
2nd - Mitochondrial fraction
3rd - Membrane fraction
4th - Cytosol fraction (Where most proteins exist)
16
Q
What are the general steps to purify a protein?
A
- Centrifugation
- Salt Out
- Dialysis
- Chromatography
17
Q
What is Salting Out?
A
Adding a salt (ammonium sulfate) to an aqueous protein solution and the salt molecules will bind the H2O molecules, freeing the protein of interest.
18
Q
What is Dialysis?
A
Place (salt + protein) in a semi-permeable tube and place tube in a buffer solution. The salt will diffuse out, leaving protein of interest in tube.
*Note: It’s more effective to use several rounds small volume than one round large volume.
19
Q
What are the various parts of an antibody? Label!
A
- antigen binding site
- light chain
- heavy chain
- variable domain
- constant domain
- disulfide bonds
20
Q
How does column chromatography work?
A
- Separates proteins based on different physical/chemical interactions with a solid gel matrix.
- Proteins that interact poorly with the matrix are eluted first from the column.
21
Q
How does gel-filtration chromatography work?
A
- Separates proteins based on size
- Large molecules elute first because they cannot fit through pores
- Smaller molecules elute last because they interact with beads
22
Q
How does High-Performance Liquid Chromatography (HPLC) work?
A
- Separates proteins based on size (same as gel-filtration)
- Contains smaller bead particles leading to better separation
Requires Applied Pressure
23
Q
How does Ion-Exchange Chromatography work?
A
- Separates proteins based on charge
- Positively charged anion matrix Diethylaminoethyl (DEAE)
- Negatively charged cation matrix Carboxyl methyl cellulose (CMC)
24
Q
How does Affinity Chromatography work?
A
- Ligand is covalently linked to beads and proteins stick to the ligand
25
What does SDS do to proteins?
Coats proteins with a _blanket negative charge._ Once the charge is equal, proteins can be separated based on size.
26
How does Polyacrylamide Gel Electrophoresis (PAGE) work?
SDS coats proteins with a uniform negative charge. Once the charge is equal, proteins can be separated based on **size**.
\*Heavier proteins move slowly and accumulate on top
27
How does the % of the polyacrylamide gel affect small and large proteins?
_Large proteins_ are best separated by SDS-PAGE using _low-percentage_ polyacrylamide gels,
-_Small proteins_ resolve better in _high-percentage_ polyacrylamide gels.
28
How does Isoelectric Focusing (IEF) work?
Separates proteins based on **pI** **value**.
Proteins migrate until they have no net charge.
29
What is 2D Gel Electrophoresis?
IEF + SDS-PAGE
Separates proteins based on **size** and **pI** **value**.
30
Each B-cell makes a single type of \_\_\_\_\_\_\_\_.
antibody
31
What is an epitope?
A specific site on an antigen where an antibody can bind to.
32
Compare monoclonal vs polyclonal antibodies.
**Monoclonal** - _Homogenous_ Ig species that recognizes _one epitope_ on an antigen
**Polyclonal** - _Heterogenous_ mixture of Ig proteins that can recognize _one or more epitopes_ on an antigen
33
How would you generate antigen-specific polyclonal antibodies?
1. Immunize rabbit with an antigen
2. Purify with affinity chromatography
34
What is a hybridoma cell?
B-cell + immortalized tumor cell
35
How would you generate monoclonal antibodies?
1. Immunize mouse and isolate antibody producing B-cells
2. Create **hybridoma** cell (B-cell + immortalized tumor cell)
3. Once a hybridoma clone is identified that secretes an antigen-specific antibody, it can be expanded in culture and used to make an **unlimited supply of antibody**.
36
What is Western Blotting used for and how does it work?
Used to identify proteins that have been separated by SDS-PAGE
1. Transfer proteins from gel to filter
2. Add primary antibody 1ºAb (protein-specific)
3. Add secondary antibody 2ºAb (1ºAb specific)
4. Add enzyme which identifies the location of the target protein
37
How does epitope tagging work?
FLAG - adds to N-terminus
myc - addes to C-terminus
Add FLAG or myc epitope sequences to a protein so that protein can later be identified using Western Blot
38
What is Enzyme-Linked Immunosorbent Assay (ELISA)?
The capture antibody is fixed to the plate and later exposed to the sample so the antigen can bind to antibody. The antigen, in turn, is bound by an additional (detection) antibody. Detection is accomplished by an enzyme-linked secondary antibody.
39
What is Immunoprecipitation?
Immunoprecipitation is a variation of affinity purification in which a **monoclonal antibody is** **covalently linked to a carbohydrate bead**. The bead is easily precipitated by gentle centrifugation, isolating the capture antigen protein. This technique is often coupled to another method, including Western blot or mass spectrometry.
40
How does Edman's Degradation provide a proteins sequence?
1. Add PITC - binds to N-terminus
2. Add TFA acid - cleaves off terminal amino acid
3. Repeat and you get 1 amino acid at a time
\*Limited to polypeptides up to 50 residues long\*
41
For proteins longer than 50 residues, enzymatic cleavage with ________ and \_\_\_\_\_\_\_\_\_is performed.
**Trypsin** - Cleaves at C-terminus of Lys and Arg
**Chymotrypsin** - Cleaves at C-terminus of Tyr, Trp, and Phe
42
What does Mass Spectrometry?
**Mass-to-charge ratio** (m/z)
\*Ionize iva **ESI** or **MALDI** - generates + ions
43
Compare ESI and MALDI peptide ionization methods.
ESI - **high voltage, evaporates solvent**
MALDI - Proteins attached to **solid matrix** and exposed to **laser**
\*Both are ionized
44
A red spot is associated with ____ dye and indicates the protein WAS affected.
Cy5
45
A green spot is associated with ____ dye and indicates the protein WAS NOT affected.
Cy3
46
For Affinity Chromatography, how would you displace the target protein?
Eluting the target protein from the affinity column involves either _adding large amounts of a competing ligand_ to the elution buffer or disrupting the binding interaction with _changes in salt or pH_.
47
The Edman degradation uses _________ to label the N-terminal protein.
PITC
48
Adding a phosphate group will ________ the charge at the pH at which the protein will be isoelectrically neutral (pI value).
decrease
49
How does Edman Degredation work?
1. The peptide is labeled at the **N terminus** with **PITC**
2. Terminal amino acid is removed with **TFA** (acid).
3. After organic extraction, the modified amino acid identity is determined using appropriate standards on paper **chromatography**.
\*Limitation: Only works for up to 50aa chain
50
How does Solid Phase Peptide Synthesis work (SPPS) work?
1. C-terminal of **AA1 is attached to resin** molecule and N-terminal protecting group **(Fmoc) is removed**
2. Activate carboxyl group of AA2 using DCC
3. AA1 + AA2 **join**
4. Remove protecting groups
5. Treat with HF to separte from resin
51
Which amino acid presents a problem for Fmoc blocking during solid state peptide synthesis?
lysine
52
What are 2 ways to determine a proteins structure?
**NMR**
**X-Ray Crystallography** - Beam of X-rays directed at a protein _crystal_ and forms a map of the **electron density.**
53
Which method is used for finding the existence of specific antibodies?
ELISA
54
The FLAG and myc epitope sequences are characterized which polar amino acid residues?
**glutamatic acid (E)** and **aspartic acid (D)**
55
Gels with less cross-linked acrylamide (low % SDS gels) will do what to the different sized proteins?
Favor the _separation of larger proteins_ at the expense of smaller ones.
56
What are 3 requirements for X-ray Crystallography?
- **95% pure** protein
- **Crystal** sturcture
- **15mg/mL** concentration
57
How do you measure protein efficiency of a target protein?
58
Which amino acids are positively charged?
Histidine **H**
Lysine **K**
Arginine **R**
59
Which amino acids are negatively charged?
Aspartic acid **D**
Glutamic acid **E**
60
61
What do metabolic enzymes do and what is an example of one?`
- **Lower activation energy**
- Increase rate of product formation
- DOES NOT alter equilibrium concentration
Ex. **Maltate dehydrogenase** - oxidizes _maltate → oxaloacetate_
62
What do structural proteins do and what are some examples?
- Maintain integrity and shape of cell; motility; cell signaling
Ex. Actin, tubulin, collagen
63
Subunits of Actin self-assemble from actin monomers and form long polymers called \_\_\_\_\_\_\_\_\_\_\_.
thin filaments
64
Tubulin self-assembles from tubulin monomers and form long polymers called \_\_\_\_\_\_\_\_\_\_\_\_.
microtubules
65
What structural protein is a primary component of connective tissue?
collagen
66
What are 3 types of membrane receptors?
**G-protein coupled receptors** - adrenergic receptors (epinephrine ligands)
**Receptor tyrosine kinases** - insulin
**Growh hormone receptors**
67
What happens when the erythropoietin hormone binds to its receptor?
Signal transduction occurs casuing cell to produce more **hemoglobin** and **erythrocytes** (RBC)
68
What happens when a ligand binds to a nuclear receptor?
_Transcription factors_ that regulate _gene expression_ in response to ligand binding.
Include steroid receptors such as Estrogen and Progesterone
69
What do protein kinases do?
Reversibly _phosphorylate_ proteins at _Ser_ and _Thr_ amino acid residues.
70
Which genomic caretaker protein combines newly formed DNA fragments?
DNA ligase
71
Which genomic caretaker protein unwinds DNA by relieving positive supercoils?
Topoisomerase
72
Which genomic caretaker protein assists DNA polymerase by preparing (priming) DNA template strand for replication?
DNA primase
73
Which genomic caretaker protein repairs thymine dimers that result from UV radiation?
Photolyase
74
Which genomic caretaker protein uses DNA template to guide the synthesis of new DNA strands?
DNA polymerase
75
What does RNA polymerase do and what are the 3 types?
Facilitates **RNA transcription** from DNA template strand
Type I - ribosomal RNA
Type II - messenger RNA
Type III - tRNA
76
How does RecBCD + RecA repair DNA?
**RecBCD** Finds defect location and cleaves it off
**RecA** Repairs the gap
77
What happens when a ligand binds to a protein?
**Conformational change** occurs which is key to protein function
78
What does albumin do?
Transports **hydrophobic** metabolites (fatty acids) through aqueous environments (cytosol, circulatory system).
79
Compare myoclobin and hemoglobin.
**Myoglobin** - Found in _muscles_, stores O2
**Hemoglobin** - _Transports_ O2 from lungs to muscles/tissues and back to lungs
\*Both proteins contain heme and bind O2 _reversibly to Fe2+_
80
Why is heme necessary in globin proteins?
Because no amino acid side chain can _bind reversibly to O2_
81
What is the heme group?
A **Fe2+-porphyrin complex** (porphyrin is the organic atoms) that functions as a _prostheitc group_ for O2 to bind to.
82
What is the structure of hemoglobin?
**Four ploypeptide subunits (2a, 2ß), 4 heme groups**
Subunits each consist of **eight α helices** with a single molecule of heme bound by each polypeptide
83
What is the structure of myoglobin?
**Single polypeptide chain, 1 heme group**
Subunits each consist of **eight α helices** with a single molecule of heme bound by each polypeptide.
84
Where does O2 bind on the heme group?
**6th coordination bond of Fe2+**
Oxygen will only bind to this prosthetic group when the iron is in the +2 oxidation state (ferrous).
85
Describe the process of oxygen binding to a heme group on either hemoglobin or myoglobin.
1. The proximal histidine (His F8) binds to Fe2+ directly
2. O2 is bound to Fe2+ at its 6th coordination bond
3. The distal histidine (His E7) forms a H-bond with the O2
Forms **oxyhemoglobin**
86
What are the locations of the proximal and distal histidine residues that bind to O2?
Proximal - His F8
Distal - His E7
The eight α helices of globin proteins are named A through H, with the proximal His residue referred to as His F8 because it is the eighth residue on the F helix. Similarly, the distal His residue is denoted as His E7 because it is the seventh residue on the E helix.
87
What is the shape of the heme group when O2 is bound and when it isn't?
**Oxy** - heme group is **planar (Relaxed, R)**
**Deoxy** - heme group is **puckered** becuase of the large Fe atom **(Tense, T)**
88
What is the concerted models that explains the cooperative binding behavior of hemoglobin?
T and R states are in **equilibrium**. T state is favored when no ligand (O2) is bound. _Binding of additional O2 molecules causes R state to be favored_
89
What is the Sequential model for explaining the cooperative behavior of hemoglobin?
When 1 O2 molecule binds to 1 subunit of hemoglobin, this induces adjacent subunits to bind to O2
90
What is fractional saturation Ø?
[P] = concentration of protein
[L] = concentration of ligand
[PL] = concentration of protein-ligand complex
91
What is Kd and how does it relate to hemoglobin/myoglobin - O2 binding?
Larger Kd value indicates more of the dissociated species is present and there is a **low affinity between hemoglobin and O2**
92
Kd is the point on a Fractional Saturation Plot at which Ø = \_\_\_\_\_\_\_.
0.5
[O2] = 0.5
93
A higher Kd indicates a _______ ligand/protein affinity.
lower
94
A lower Kd indicates a _______ ligand/protein affinity.
higher
95
What does the Oxygen Binding Curve for myglobin look like?
Hyperbolic
About _20% bound O2 is released_ by myoglobin when muscle is _active_
96
What does the Oxygen Binding Curve for hemoglobin look like?
_Sigmoidal_ due to _cooperative binding_
About 60% O2 is released when transported from lungs to tissue
97
Ligand binding is a reversible process involving _________ interactions.
noncovalent
98
What are effector molecules?
Allosteric molecules which can **increase (positive)** or **decrease (negative)** the affinity a ligand has for a substrate.
99
When using partial pressure as in pO2, the half-saturation value is referred to as _____ instead of Kd
P50
100
What is cooperative binding as it relates to hemoglobin and O2?
The binding of the first O2 to the protein complex facilitates the binding of additional O2 molecules on the same protein.
101
What are the 3 negative allosteric effectors that affect O2 binding to hemoglobin?
1. **pH, [H+}**: As [H+} increases, O2 affinity decreases
2. **CO2**: As [CO2} increases, O2 affinity decreases
3. **2,3-BPG**: Binding of 2,3-BPG to hemoglobin decreases O2 affinity for hemoglobin
102
Why is Oxygen considered a positive allosteric effector of hemoglobin?
The binding of a single O2 molecule to one globin subunit shifts the conformational equilibrium from the T-state toward the R-state. This _increases O2 affinity in other subunits_
103
Where does CO2 bind in order to behave as a negative allosteric effector?
Binds to the **N-terminus** of the polypeptide chain
104
What is the Bohr Effect?
Combination of low **pH** and high **[CO2]**
Increased [CO2} also causes more H+ to be produced providing a _double negative allosteric effector_ for O2 binding to hemoglobin
105
Where does 2,3-BPG bind in order to behave as a negative allosteric effector?
Between the ß-subunits at the center of hemoglobin preventing O2 from binding to heme group.
106
When does 2,3-BPG bind to hemoglobin?
In the **tissues**, the combination of reduced O2 levels and the Bohr Effect favor the T-state conformation, which is _stabilized by 2,3-BPG binding_
107
Why does fetal hemoglobin have a higher affinity for O2 than maternal hemoglobin?
Fetus has α2γ2 subunits instead of α2β2 subunits. The **2 γ subunits are less positive** and therefore do not strongly attract 2,3-BPG (a highly negative molecule).
108
Compare homotropic allostery vs heterotropic allostery.
**Homotropic** - O2 binds to primary site (heme group) increasing Hb affinity of other O2 molecules
**Heterotropic -** H+, CO2, and 2,3-BPG bind to _secondary sites_ which affects O2 binding to the primary site (heme group)
109
Which histidine residues are conserved in all three proteins (a, B, myoglobin) because they are the amino acids required to coordinate the oxygen binding in the heme cofactor of the given globin molecules?
Histidines at positions **E7** and **F8**
110
What is a conserved amino acid?
One that shows up at the **same position in multiple protein** subunits.
Mutating a highly conserved amino acid to a vastly different amino acid is likely to _affect the correct structure and function_ of a protein.
111
Which molecule decreases hemoglobin's affinity for oxygen most drastically?
**Carbon monoxide** binds to the same site on hemoglobin as oxygen but with a higher affinity. This causes hemoglobin bound by carbon monoxide to have a lower affinity for oxygen.
112
What causes Sickle Cell Anemia?
_EV6 Substitution_
**Glutamate** is substituted for **Valine** at position 6 in the ß-globin polypeptide.
113
Fetal hemoglobin has a _______ affinity for 2,3-BPG than maternal hemoglobin due to its altered subunit. As a result, more hemoglobin molecules are in the _________ in fetal hemoglobin
lower
R-state
114
Phospholipids have 2 hydrocarbon tails and self-organize into __________ while fatty acids have 1 hydrocarbon tail and self-organize into \_\_\_\_\_\_\_\_\_.
bilayers
micelles
115
What are the 3 types of membrane proteins?
**Integral** - Non-polar and embedded inside bilayer, Transporters
**Peripheral** - Polar and located on the surface, Receptors/enzymes
**Amphitropic** - Polar and non-polar, Palmitoylated proteins (reversible)
116
What are the two components of phospholipids?
**2 fatty acid tails** and a **phosphate group** attached to glycerol.
Basically, you take a triglyceride and substitute one fatty acid tail for a phosphate group.
117
What is palmitoylation and on which aa residue does it occur?
Adding of palmitic acid (**fatty acid**) to -SH group on _Cysteine_
118
How do hydrophobic and hydrophilic molecules move across the membrane?
Hydrophobic **diffuse** down their concentration gradient into the cell.
Hydrophilic molecules require **passive transport** down their concentration gradient (NO ATP REQUIRED).
**Active transport** moves molecules _against_ their concentration gradient (ATP REQUIRED)
119
What is the formula for Gibbs Free Energy used to calculate molecules movement across a membrane? Constant values?
R = 8.314 J/mol•k
Z = charge (Na is +1)
F = 96500F
V = -70mV resting membrane potential
120
Cells maintain a _______ charge inside the cell in reference to the outside. What is the resting membrane potential?
negative
Resting membrane potential = -70mV
121
Compare channels and pumps.
Channels: [High] → [Low] passive
Pumps: [Low] → [High] active
122
What is a mitochondrial voltage-dependent anion channel (VDAC)?
A passive membrane transporter in the porin family. Has a **ß-Barrell structure** with **alternating polar/nonpolar** aa residues with hydrophobic nonpolar residues oriented mostly outward (valine, leucine, isoleucine) and hydrophilic and charged groups oriented mostly inward (serine, threonine, arginine, lysine, aspartate).
123
What are aquaporins?
A major class of _passive membrane transport_ proteins that transport **water** molecules across a hydrophobic membrane but can also transport **urea** and **glycerol.**
124
What is the K+ channel?
A passive ion channel that **moves K+ out of the cell.** H2O facilitates the movement of the potassium ions _so they don't repel_ each other.
125
Where are the two places that energy for active transport comes from?
- ATP hydrolysis
- Ionic gradient
126
How does action potential work?
1. Resting potential (-70mV)
2. Signal received
3. Na+ channel opens and Na+ ions enter cell (depolarization, +40mV)
4. K+ channel opens and K+ ions leave cell (repolarization)
5. Hyperpolarization occurs where the membrane potential goes below the initial resting potential
6. Resting potential
127
Primary active transport is ______ dependent and secondary active transport utilizes an ___________ gradient.
ATP
electrochemical
128
Compare the 3 types of active transport.
**Uniport** - Uses **ATP** to transport from [Low] → [High]
**Symport** - Moves one molecule from [High] → [Low] and the energy given off is used to move a different molecule from [Low] → [High]. Transport occurs in **same** **direction**
**Antiport** - Same a symport except transport is in **opposite direction**
129
What is the Na+-K+ ATPase membrane protein?
The Na+–K+ ATPase membrane protein is a **P-type primary active transporter.** It moves 3 Na+ ions out of the cell and 2 K+ ions into the cell. ATP phosphorylates the transport protein leading to a conformational change allowing it to move the ions against their electrochemical gradients.
130
What are the two types of primary active transport?
**P-type**: phosphorylates
**ABC**: ATP binding cassette
131
The ABC transporter mechanism utilizes 2 ______ molecules. What are the steps?
ATP
132
What is the bacterial lactose permease transporter? What does it move into the cell?
A _secondary active symporter_ that utilizes a steep **H+ gradient** to move in **lactose** against its concentration gradient.
133
What is the Human Na+- I- symporter?
A _secondary active symporter_ that utilizes a Na+ gradient to move in I- against its concentration gradient.
134
ATP hydrolysis generates how much energy?
**ΔG = -7.3 kcal/mol**
135
1 kCal/mol = ___ kJ/mol
4.2
136
How does an enzyme lower the activation energy of a reaction?
By forming an **ES\* complex** which has a high free energy (ΔG)
137
Enzymes can be either proteins or \_\_\_\_\_\_\_\_.
**RNA**
e.g. Ribozyme
138
What is catalytic power?
139
Compare the lock and key model to the induced fit model.
**Lock and key** - Substrate binds to enzyme **perfectly**
**Induced fit** - _Enzyme is flexible_ to accommodate many substrates
140
Compare Cofactors, Coenzymes, and Prosthetic groups.
**Cofactors** - _Small molecules_ that aid in the catalytic reaction of enzymes. Include ions such as Fe2+, Cu2+, and Mg2+
**Coenzymes** - Cofactors with _organic_ components. Includes vitamin derived species such as NAD+ and FAD+. Also known as co-substrates
**Prosthetic group** - Coenzymes that are _permanently associated_ with enzymes
DOES NOT INCLUDE AMINO ACID RESIDUES
141
Compare apoenzymes and haloenzymes.
Apoenzyme (inactive) + cofactor = Holoenzyme (active)
142
DNase is inactive until _____ is added forming __________ which cleaves phosphodiester bonds turning DNA into nucleotides.
Mg2+
DNase-Mg2+
_EDTA binds to Mg2+_ which keeps it from being a cofactor for DNase
143
Enzymes can be inactivated by depletion of \_\_\_\_\_\_\_\_\_.
cofactors
EDTA binds to Mg2+ which keeps it from being a cofactor for DNase
144
Which class of enzyme is responsible for oxidation-reduction reactions (transfer of H or O atoms)?
Oxidoreductase
Oxidases, dehydrogenases
145
Which class of enzyme is responsible for the transfer of functional groups?
Transferase
Kinases, transaminases
146
Which class of enzyme is responsible for hydrolyzing a substrate forming 2 products?
Hydrolase
Peptidases, lipases
147
Which class of enzyme is responsible for the cleavage of C-C, C-O, or C-N bonds by means other than hydrolysis or oxidation?
Lyase
Decarboxylases, carboxylases
148
Which class of enzyme is responsible for intramolecular rearrangements?
Isomerases
Mutases
149
Which class of enzyme is responsible for the formation of C-C, C-O, C-S, or C-N bonds using ATP cleavage?
Ligase
Synthetases
150
What is constant during Steady-State Conditions of an enzyme reaction?
**[ES] and [E]**
[S] and [P] increases/decrease linearly
151
What is the Michaelis-Menten equation?
152
What is Km and what is the equation?
[Substrate] at 1/2Vmax
153
What does a low and high Km value signify?
**Lower Km**: Higher enzyme/substrate affinity
**Higher Km**: Lower enzyme/substrate affinity
154
What is the Lineweaver-Burk Double Reciprocal Equation?
y = mx + b format
155
What is the International unit (IU) of enzyme activity?
micromoles/minute
156
What is the Specific unit of an enzyme?
micromoles/min • mg protein
157
What is the turnover number (Kcat)?
Amount of substrate molecules converted into product by one enzyme per second.
Kcat = Vmax/[Et]
158
What is catalytic efficiency (specificity constant)?
Kcat/Km
A "perfect" enzyme has a large Kcat and small Km
159
What are 3 types of Enzyme Inhibition?
**Reversible** - _non-covalent bonding_ (competitive, uncompetitive, non-competitive)
**Irreversible** - _covalent bonding_ "kills" enzyme by binding tightly
**Inhibition/depletion of cofactors**
160
What is competitive inhibition? What is the Km equation you would use if competitive inhibitor was present?
A molecule that binds to the same site (active site) as the substrate.
- Can be overcome by increasing [S].
161
Compare how the 4 types of inhibitors affect Vmax and Km.
**Competitive**: Vmax unchanged, Km increases
**Uncompetitive**: Vmax decreased, Km decreased
**Mixed type**: Vmax decreased, Km increase or decrease
**Noncompetitive**: Vmax decreased, Km unchanged
162
What do the kinetics graphs look like for a competitive inhibitor?
163
What is Uncompetitive Inhibition?
Inhibitor binds to the _enzyme-substrate complex_ and increases the affinity for the enzyme towards its substrate, making a highly stable compound (no product formed)
164
Compare how the 4 types of inhibitors bind.
**Competitive**: Binds to free enzyme
**Uncompetitive**: Binds to enzyme-substrate complex
**Mixed type**: Binds to free enzyme or enzyme-substrate complex
**Noncompetitive**: Same as Mixed type; K1 = K'1
165
What do the kinetics graphs look like for an uncompetitive inhibitor?
166
What do the kinetics graphs look like for a mixed type inhibitor?
167
What is irreversbile inhibition? What do the kinetics graphs look like for irreversible inhibitor?
Inhibitor forms a _covalent bond_ in the active site of an ezyme on Ser or Cys residues. Similar to noncompetitive
168
What are the 2 main enzyme regulatory mechanisms?
**Bioavailability** - protein synthesis, enzyme degredation
**Catalytic efficiency** - affinity of enzyme toward substrate
169
Compare homotropic and heterotropic effectors.
**Homotropic** effectors – substrate itself
**Heterotropic** effectors - other than substrate
170
The most common modification is phosphorylation of \_\_\_\_, \_\_\_\_\_, and _____ residues by kinases.
Ser, Thr, and Tyr
Phosphatases remove phosphate groups
171
What is the formula for the alpha factor in relation to enzyme kinetics?
