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CHEM 441A > Midterm 2 > Flashcards

Midterm 2 Flashcards

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1
Q

What type of chaperone is GroEL–GroES?

A

chamber

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2
Q

Many diseases, including Alzheimer’s disease, are associated with

A

protein aggregation

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3
Q

Globular proteins are mostly ________ and fibrous proteins are mostly _________.

A

soluble

insoluble

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4
Q

Which fibrous protein is a myosin coiled-coil protein?

A

keratin

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5
Q

Describle keratin’s structure.

A

Left-handed coiled coil dimer

Right-handed helix

Disulfide bridges between 2 coiled coils

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6
Q

Which fibrous protein is the aa sequence (G-A-S) repeated and tightly packed?

A

Silk

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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.

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8
Q

Which fibrous protein is (HyPro-Pro-Gly) repeated?

A

Collagen

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9
Q

Protein folding is a ________ process.

A

spontaneous (exothermic)

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10
Q

What are chaperone proteins and what 3 functions of chaperones?

A

Proteins that facilitate the formation of stable 3D sturctures.

  1. Help proteins fold properly
  2. Fix misfolded proteins
  3. Disrupt aggregates
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11
Q

What are the 2 types of chaperone proteins?

A
  1. Clamp - heat shock
  2. Chamber -GroEL–GroES protein complex
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12
Q

What are the 3 Models of Protein Folding?

A
  1. Hydrophobic collapse - clustering of hydrophobic side chains
  2. Framework model - 2º/3º structures form independently
  3. Nucleation model - localized interactions between 2º stuctures
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13
Q

What are the 4 general steps involved in protein study?

A
  • Cloning
  • Expression
  • Purification
  • Characterization
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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

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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)

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16
Q

What are the general steps to purify a protein?

A
  1. Centrifugation
  2. Salt Out
  3. Dialysis
  4. Chromatography
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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.

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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.

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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
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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.
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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
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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

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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)
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24
Q

How does Affinity Chromatography work?

A
  • Ligand is covalently linked to beads and proteins stick to the ligand
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25
Q

What does SDS do to proteins?

A

Coats proteins with a blanket negative charge. Once the charge is equal, proteins can be separated based on size.

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26
Q

How does Polyacrylamide Gel Electrophoresis (PAGE) work?

A

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

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27
Q

How does the % of the polyacrylamide gel affect small and large proteins?

A

Large proteins are best separated by SDS-PAGE using low-percentage polyacrylamide gels,

-Small proteins resolve better in high-percentage polyacrylamide gels.

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28
Q

How does Isoelectric Focusing (IEF) work?

A

Separates proteins based on pI value.

Proteins migrate until they have no net charge.

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29
Q

What is 2D Gel Electrophoresis?

A

IEF + SDS-PAGE

Separates proteins based on size and pI value.

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30
Q

Each B-cell makes a single type of ________.

A

antibody

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31
Q

What is an epitope?

A

A specific site on an antigen where an antibody can bind to.

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32
Q

Compare monoclonal vs polyclonal antibodies.

A

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

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33
Q

How would you generate antigen-specific polyclonal antibodies?

A
  1. Immunize rabbit with an antigen
  2. Purify with affinity chromatography
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34
Q

What is a hybridoma cell?

A

B-cell + immortalized tumor cell

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35
Q

How would you generate monoclonal antibodies?

A
  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.
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36
Q

What is Western Blotting used for and how does it work?

A

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
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37
Q

How does epitope tagging work?

A

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

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38
Q

What is Enzyme-Linked Immunosorbent Assay (ELISA)?

A

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.

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39
Q

What is Immunoprecipitation?

A

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.

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40
Q

How does Edman’s Degradation provide a proteins sequence?

A
  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*

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41
Q

For proteins longer than 50 residues, enzymatic cleavage with ________ and _________is performed.

A

Trypsin - Cleaves at C-terminus of Lys and Arg

Chymotrypsin - Cleaves at C-terminus of Tyr, Trp, and Phe

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42
Q

What does Mass Spectrometry?

A

Mass-to-charge ratio (m/z)

*Ionize iva ESI or MALDI - generates + ions

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43
Q

Compare ESI and MALDI peptide ionization methods.

A

ESI - high voltage, evaporates solvent

MALDI - Proteins attached to solid matrix and exposed to laser

*Both are ionized

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44
Q

A red spot is associated with ____ dye and indicates the protein WAS affected.

A

Cy5

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45
Q

A green spot is associated with ____ dye and indicates the protein WAS NOT affected.

A

Cy3

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46
Q

For Affinity Chromatography, how would you displace the target protein?

A

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.

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47
Q

The Edman degradation uses _________ to label the N-terminal protein.

A

PITC

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48
Q

Adding a phosphate group will ________ the charge at the pH at which the protein will be isoelectrically neutral (pI value).

A

decrease

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49
Q

How does Edman Degredation work?

A
  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

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50
Q

How does Solid Phase Peptide Synthesis work (SPPS) work?

A
  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
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51
Q

Which amino acid presents a problem for Fmoc blocking during solid state peptide synthesis?

A

lysine

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52
Q

What are 2 ways to determine a proteins structure?

A

NMR

X-Ray Crystallography - Beam of X-rays directed at a protein crystal and forms a map of the electron density.

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53
Q

Which method is used for finding the existence of specific antibodies?

A

ELISA

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54
Q

The FLAG and myc epitope sequences are characterized which polar amino acid residues?

A

glutamatic acid (E) and aspartic acid (D)

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55
Q

Gels with less cross-linked acrylamide (low % SDS gels) will do what to the different sized proteins?

A

Favor the separation of larger proteins at the expense of smaller ones.

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56
Q

What are 3 requirements for X-ray Crystallography?

A
  • 95% pure protein
  • Crystal sturcture
  • 15mg/mL concentration
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57
Q

How do you measure protein efficiency of a target protein?

A
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58
Q

Which amino acids are positively charged?

A

Histidine H

Lysine K

Arginine R

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59
Q

Which amino acids are negatively charged?

A

Aspartic acid D

Glutamic acid E

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60
Q
A
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61
Q

What do metabolic enzymes do and what is an example of one?`

A
  • Lower activation energy
  • Increase rate of product formation
  • DOES NOT alter equilibrium concentration

Ex. Maltate dehydrogenase - oxidizes maltate → oxaloacetate

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62
Q

What do structural proteins do and what are some examples?

A
  • Maintain integrity and shape of cell; motility; cell signaling

Ex. Actin, tubulin, collagen

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63
Q

Subunits of Actin self-assemble from actin monomers and form long polymers called ___________.

A

thin filaments

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64
Q

Tubulin self-assembles from tubulin monomers and form long polymers called ____________.

A

microtubules

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65
Q

What structural protein is a primary component of connective tissue?

A

collagen

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66
Q

What are 3 types of membrane receptors?

A

G-protein coupled receptors - adrenergic receptors (epinephrine ligands)

Receptor tyrosine kinases - insulin

Growh hormone receptors

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67
Q

What happens when the erythropoietin hormone binds to its receptor?

A

Signal transduction occurs casuing cell to produce more hemoglobin and erythrocytes (RBC)

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68
Q

What happens when a ligand binds to a nuclear receptor?

A

Transcription factors that regulate gene expression in response to ligand binding.

Include steroid receptors such as Estrogen and Progesterone

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69
Q

What do protein kinases do?

A

Reversibly phosphorylate proteins at Ser and Thr amino acid residues.

70
Q

Which genomic caretaker protein combines newly formed DNA fragments?

A

DNA ligase

71
Q

Which genomic caretaker protein unwinds DNA by relieving positive supercoils?

A

Topoisomerase

72
Q

Which genomic caretaker protein assists DNA polymerase by preparing (priming) DNA template strand for replication?

A

DNA primase

73
Q

Which genomic caretaker protein repairs thymine dimers that result from UV radiation?

A

Photolyase

74
Q

Which genomic caretaker protein uses DNA template to guide the synthesis of new DNA strands?

A

DNA polymerase

75
Q

What does RNA polymerase do and what are the 3 types?

A

Facilitates RNA transcription from DNA template strand

Type I - ribosomal RNA

Type II - messenger RNA

Type III - tRNA

76
Q

How does RecBCD + RecA repair DNA?

A

RecBCD Finds defect location and cleaves it off

RecA Repairs the gap

77
Q

What happens when a ligand binds to a protein?

A

Conformational change occurs which is key to protein function

78
Q

What does albumin do?

A

Transports hydrophobic metabolites (fatty acids) through aqueous environments (cytosol, circulatory system).

79
Q

Compare myoclobin and hemoglobin.

A

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
Q

Why is heme necessary in globin proteins?

A

Because no amino acid side chain can bind reversibly to O2

81
Q

What is the heme group?

A

A Fe2+-porphyrin complex (porphyrin is the organic atoms) that functions as a prostheitc group for O2 to bind to.

82
Q

What is the structure of hemoglobin?

A

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
Q

What is the structure of myoglobin?

A

Single polypeptide chain, 1 heme group

Subunits each consist of eight α helices with a single molecule of heme bound by each polypeptide.

84
Q

Where does O2 bind on the heme group?

A

6th coordination bond of Fe2+

Oxygen will only bind to this prosthetic group when the iron is in the +2 oxidation state (ferrous).

85
Q

Describe the process of oxygen binding to a heme group on either hemoglobin or myoglobin.

A
  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
Q

What are the locations of the proximal and distal histidine residues that bind to O2?

A

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
Q

What is the shape of the heme group when O2 is bound and when it isn’t?

A

Oxy - heme group is planar (Relaxed, R)

Deoxy - heme group is puckered becuase of the large Fe atom (Tense, T)

88
Q

What is the concerted models that explains the cooperative binding behavior of hemoglobin?

A

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
Q

What is the Sequential model for explaining the cooperative behavior of hemoglobin?

A

When 1 O2 molecule binds to 1 subunit of hemoglobin, this induces adjacent subunits to bind to O2

90
Q

What is fractional saturation Ø?

A

[P] = concentration of protein

[L] = concentration of ligand

[PL] = concentration of protein-ligand complex

91
Q

What is Kd and how does it relate to hemoglobin/myoglobin - O2 binding?

A

Larger Kd value indicates more of the dissociated species is present and there is a low affinity between hemoglobin and O2

92
Q

Kd is the point on a Fractional Saturation Plot at which Ø = _______.

A

0.5

[O2] = 0.5

93
Q

A higher Kd indicates a _______ ligand/protein affinity.

A

lower

94
Q

A lower Kd indicates a _______ ligand/protein affinity.

A

higher

95
Q

What does the Oxygen Binding Curve for myglobin look like?

A

Hyperbolic

About 20% bound O2 is released by myoglobin when muscle is active

96
Q

What does the Oxygen Binding Curve for hemoglobin look like?

A

Sigmoidal due to cooperative binding

About 60% O2 is released when transported from lungs to tissue

97
Q

Ligand binding is a reversible process involving _________ interactions.

A

noncovalent

98
Q

What are effector molecules?

A

Allosteric molecules which can increase (positive) or decrease (negative) the affinity a ligand has for a substrate.

99
Q

When using partial pressure as in pO2, the half-saturation value is referred to as _____ instead of Kd

A

P50

100
Q

What is cooperative binding as it relates to hemoglobin and O2?

A

The binding of the first O2 to the protein complex facilitates the binding of additional O2 molecules on the same protein.

101
Q

What are the 3 negative allosteric effectors that affect O2 binding to hemoglobin?

A
  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
Q

Why is Oxygen considered a positive allosteric effector of hemoglobin?

A

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
Q

Where does CO2 bind in order to behave as a negative allosteric effector?

A

Binds to the N-terminus of the polypeptide chain

104
Q

What is the Bohr Effect?

A

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
Q

Where does 2,3-BPG bind in order to behave as a negative allosteric effector?

A

Between the ß-subunits at the center of hemoglobin preventing O2 from binding to heme group.

106
Q

When does 2,3-BPG bind to hemoglobin?

A

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
Q

Why does fetal hemoglobin have a higher affinity for O2 than maternal hemoglobin?

A

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
Q

Compare homotropic allostery vs heterotropic allostery.

A

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
Q

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?

A

Histidines at positions E7 and F8

110
Q

What is a conserved amino acid?

A

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
Q

Which molecule decreases hemoglobin’s affinity for oxygen most drastically?

A

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
Q

What causes Sickle Cell Anemia?

A

EV6 Substitution

Glutamate is substituted for Valine at position 6 in the ß-globin polypeptide.

113
Q

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

A

lower

R-state

114
Q

Phospholipids have 2 hydrocarbon tails and self-organize into __________ while fatty acids have 1 hydrocarbon tail and self-organize into _________.

A

bilayers

micelles

115
Q

What are the 3 types of membrane proteins?

A

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
Q

What are the two components of phospholipids?

A

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
Q

What is palmitoylation and on which aa residue does it occur?

A

Adding of palmitic acid (fatty acid) to -SH group on Cysteine

118
Q

How do hydrophobic and hydrophilic molecules move across the membrane?

A

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
Q

What is the formula for Gibbs Free Energy used to calculate molecules movement across a membrane? Constant values?

A

R = 8.314 J/mol•k

Z = charge (Na is +1)

F = 96500F

V = -70mV resting membrane potential

120
Q

Cells maintain a _______ charge inside the cell in reference to the outside. What is the resting membrane potential?

A

negative

Resting membrane potential = -70mV

121
Q

Compare channels and pumps.

A

Channels: [High] → [Low] passive

Pumps: [Low] → [High] active

122
Q

What is a mitochondrial voltage-dependent anion channel (VDAC)?

A

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
Q

What are aquaporins?

A

A major class of passive membrane transport proteins that transport water molecules across a hydrophobic membrane but can also transport urea and glycerol.

124
Q

What is the K+ channel?

A

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
Q

Where are the two places that energy for active transport comes from?

A
  • ATP hydrolysis
  • Ionic gradient
126
Q

How does action potential work?

A
  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
Q

Primary active transport is ______ dependent and secondary active transport utilizes an ___________ gradient.

A

ATP

electrochemical

128
Q

Compare the 3 types of active transport.

A

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
Q

What is the Na+-K+ ATPase membrane protein?

A

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
Q

What are the two types of primary active transport?

A

P-type: phosphorylates

ABC: ATP binding cassette

131
Q

The ABC transporter mechanism utilizes 2 ______ molecules. What are the steps?

A

ATP

132
Q

What is the bacterial lactose permease transporter? What does it move into the cell?

A

A secondary active symporter that utilizes a steep H+ gradient to move in lactose against its concentration gradient.

133
Q

What is the Human Na+- I- symporter?

A

A secondary active symporter that utilizes a Na+ gradient to move in I- against its concentration gradient.

134
Q

ATP hydrolysis generates how much energy?

A

ΔG = -7.3 kcal/mol

135
Q

1 kCal/mol = ___ kJ/mol

A

4.2

136
Q

How does an enzyme lower the activation energy of a reaction?

A

By forming an ES* complex which has a high free energy (ΔG)

137
Q

Enzymes can be either proteins or ________.

A

RNA

e.g. Ribozyme

138
Q

What is catalytic power?

A
139
Q

Compare the lock and key model to the induced fit model.

A

Lock and key - Substrate binds to enzyme perfectly

Induced fit - Enzyme is flexible to accommodate many substrates

140
Q

Compare Cofactors, Coenzymes, and Prosthetic groups.

A

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
Q

Compare apoenzymes and haloenzymes.

A

Apoenzyme (inactive) + cofactor = Holoenzyme (active)

142
Q

DNase is inactive until _____ is added forming __________ which cleaves phosphodiester bonds turning DNA into nucleotides.

A

Mg2+

DNase-Mg2+

EDTA binds to Mg2+ which keeps it from being a cofactor for DNase

143
Q

Enzymes can be inactivated by depletion of _________.

A

cofactors

EDTA binds to Mg2+ which keeps it from being a cofactor for DNase

144
Q

Which class of enzyme is responsible for oxidation-reduction reactions (transfer of H or O atoms)?

A

Oxidoreductase

Oxidases, dehydrogenases

145
Q

Which class of enzyme is responsible for the transfer of functional groups?

A

Transferase

Kinases, transaminases

146
Q

Which class of enzyme is responsible for hydrolyzing a substrate forming 2 products?

A

Hydrolase

Peptidases, lipases

147
Q

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?

A

Lyase

Decarboxylases, carboxylases

148
Q

Which class of enzyme is responsible for intramolecular rearrangements?

A

Isomerases

Mutases

149
Q

Which class of enzyme is responsible for the formation of C-C, C-O, C-S, or C-N bonds using ATP cleavage?

A

Ligase

Synthetases

150
Q

What is constant during Steady-State Conditions of an enzyme reaction?

A

[ES] and [E]

[S] and [P] increases/decrease linearly

151
Q

What is the Michaelis-Menten equation?

A
152
Q

What is Km and what is the equation?

A

[Substrate] at 1/2Vmax

153
Q

What does a low and high Km value signify?

A

Lower Km: Higher enzyme/substrate affinity

Higher Km: Lower enzyme/substrate affinity

154
Q

What is the Lineweaver-Burk Double Reciprocal Equation?

A

y = mx + b format

155
Q

What is the International unit (IU) of enzyme activity?

A

micromoles/minute

156
Q

What is the Specific unit of an enzyme?

A

micromoles/min • mg protein

157
Q

What is the turnover number (Kcat)?

A

Amount of substrate molecules converted into product by one enzyme per second.

Kcat = Vmax/[Et]

158
Q

What is catalytic efficiency (specificity constant)?

A

Kcat/Km

A “perfect” enzyme has a large Kcat and small Km

159
Q

What are 3 types of Enzyme Inhibition?

A

Reversible - non-covalent bonding (competitive, uncompetitive, non-competitive)

Irreversible - covalent bonding “kills” enzyme by binding tightly

Inhibition/depletion of cofactors

160
Q

What is competitive inhibition? What is the Km equation you would use if competitive inhibitor was present?

A

A molecule that binds to the same site (active site) as the substrate.

  • Can be overcome by increasing [S].
161
Q

Compare how the 4 types of inhibitors affect Vmax and Km.

A

Competitive: Vmax unchanged, Km increases

Uncompetitive: Vmax decreased, Km decreased

Mixed type: Vmax decreased, Km increase or decrease

Noncompetitive: Vmax decreased, Km unchanged

162
Q

What do the kinetics graphs look like for a competitive inhibitor?

A
163
Q

What is Uncompetitive Inhibition?

A

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
Q

Compare how the 4 types of inhibitors bind.

A

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
Q

What do the kinetics graphs look like for an uncompetitive inhibitor?

A
166
Q

What do the kinetics graphs look like for a mixed type inhibitor?

A
167
Q

What is irreversbile inhibition? What do the kinetics graphs look like for irreversible inhibitor?

A

Inhibitor forms a covalent bond in the active site of an ezyme on Ser or Cys residues. Similar to noncompetitive

168
Q

What are the 2 main enzyme regulatory mechanisms?

A

Bioavailability - protein synthesis, enzyme degredation

Catalytic efficiency - affinity of enzyme toward substrate

169
Q

Compare homotropic and heterotropic effectors.

A

Homotropic effectors – substrate itself

Heterotropic effectors - other than substrate

170
Q

The most common modification is phosphorylation of ____, _____, and _____ residues by kinases.

A

Ser, Thr, and Tyr

Phosphatases remove phosphate groups

171
Q

What is the formula for the alpha factor in relation to enzyme kinetics?

A

CHEM 441A (4 decks)

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