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CU: Biochemistry > Exam 1 > Flashcards

Exam 1 Flashcards

1
Q

What is biochemistry?

A

The study of the chemistry of life processes
(reactions taking place in cells)

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2
Q

What are involved in biochemistry processes?

A

The interplay of large biological macromolecules (proteins, nucleic acids) and low-molecular-weight metabolites (ie glucose and glycerol)

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3
Q

What is an example of the concept of biological unity and diversity?

A

A protein may have a similar shape in three different organisms

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4
Q

What must evolutionists assume?

A

Biochemical evolution that different organisms having macromolecules with a similar structure and common biochemical processes suggests common ancestors

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5
Q

What is DNA?

A

It is a natural linear polymer that has four building blocks: sugar (deoxyribose), phosphate, and a nitrogenous base (adenine, cytosine, guanine, thymine)

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6
Q

What are the components of DNA?

A

backbone, double helix of two antiparallel strands composed of Watson-Crick Base Pairs

Double helix explains ability to store information as bases and replicate.

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7
Q

What is special about the replication of DNA?

A

Each strand of DNA is a template for the creation of a daughter strand and complementary strands form in pico to microseconds (the timescale of most biochemical interactions)

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8
Q

What is notable about covalent bonds? What is an example of these?

A

Strong bonds, distance of 1.54A 355 kJ/mol

example: holds together DNA backbone of sugar and phosphate

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9
Q

What is notable about ionic interactions?

A

They are between fully charged atoms/molecules.
The electrostatic attraction is determined by an equation (a function of how close the electrons are)
Distance: 3 A Strength 5.86 kJ/mol

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10
Q

What is notable about electric dipoles? What is an example of these?

A

AKA dipole-dipole
due to uneven electron distribution, deals with partial charges
- can interact with ions or other dipoles 4-5kJ/mol

Example: DNA phosphate and water- water’s high dielectric constant stabilizes negatively charged backbone

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11
Q

What is notable about Hydrogen bonds? What is an example?

A

Distance 1.5-2.6 A, energy: 4-20kJ/mol
H - N/O/S/Phosphorus
H-bond acceptors need lone pair of electrons

Example: DNA base pairings. H2O bonds with base until proper pair comes by
G-C two H-bonds
A-T three H-bonds
space of remaining water H bonded to base keeps improper base from getting too close

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12
Q

What is notable about van der Walls interactions?
What is an example?

A

Unlike dipole-dipole interactions that must have something polar, these interactions occur at a given moment where an atom has more electrons
-Too close repel, too far no interaction
2-4kJ/mol

Example: interior DNA bases interact (optimal distace 4 vdW) using vdW (“pie stacking” the flat aromatic rings stack on top of each other)

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13
Q

What are the general ideas of the three laws of thermodynamics?

A

1) The total energy of the surroundings and the system stays constant
2) The total entropy of the system and its surroundings always increases (disorder has to be created)
3) Entropy can decrease locally in a system if it is increased in the surroundings

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14
Q

What is Gibbs Free Energy?

A

AG = AHsys - TASsys
a state function that describes the energetics of biochemical reactions

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15
Q

How does Gibbs Free Energy influence biochemical reactions?

A

AG < 0 for spontaneous reactions (for biochemical reactions to occur)

Must either have a large -AHsys (heat released) or lots of ASsys (disorder)

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16
Q

What are the thermodynamics of DNA formation?

A

Entropy decreases but enthalpy increases by heat being released

typically released heat allows for reactions to occur

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17
Q

What is the focus of Acid-Base reactions? What characterizes Acid-Base reactions? What Acid-Base definitions are used in biochemistry?

A

Addition or removal of hydrogen ions from molecules.
Characterized by Ka = [H+][A-]/[HA]
The bronstead-lowry definition
Acid: proton (H+) donator
Base: proton (H+) acceptor

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18
Q

What is pH?

A

It is a measure of the concentration of protons
pH = -log[H+]

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19
Q

What is the hydrophobic effect?

A

The tendency of nonpolar groups to come together to minimize their interaction with water.

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20
Q

What is the equilibrium constant of water?

A

Kw = [H+][OH-]
Pure water: [H+] = [OH-] = 10^-7 = 7.0

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21
Q

Why are Acids and Bases important to biochemistry?

A

too much acid and too much base will disrupt/denature DNA
Baseness deprotonates and Acidity protonates base pairs disrupting hydrogen bonding and causing the DNA to split apart

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22
Q

What is pka? When does it equal pH?

A

The ability to give off a proton
Ka = [H+][A-]/[HA]
pka = -log(Ka)

pKa= pH when protonated acid = deprotonated acid
pKa low = easily deprotonated
pKa high = not easily deprotonated

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23
Q

What are buffers?

A

Substances that regulate pH
They are most effective at pH near their own pKa

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24
Q

What is the Henderson-Hasselbalch equation?

A

Quantitative terms for the effect of the buffer.

pH = pKa + log ([A-]/[HA])

weak acids aer most effective as buffers at a pH near the pKa value of the acid

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25
Q

What is special about phosphate buffers?

A

They are useful in biochemical processes because one of their pKa values is 7.21 which is very close to the physiological pH: 7.4

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26
Q

What is a protein?

A

A linear polymer made of monomers called amino acids with a wide range of functional groups causing the wide range of protein function.
Interact with one another and other macromolecules to create complex assemblies with additional functions
Can be flexible or rigid (i.e. ferratin that is flexible without iron and rigid with it)

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27
Q

What are the aspects of an alpha amino acid?

A
  • alpha carbon, amino acid group, carboxylic acid group, hydrogen atom, R group
    They are chiral (have handedness)
  • All are S except Cysteine
  • All in the human body are L isomers not D isomers
  • Are zwitterions
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28
Q

What is an alpha carbon?

A

Carbon adjacent to a carbonyl (C=O)

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29
Q

What is a zwitterion?

A

A molecule that has a positive and negative charge at the same time

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30
Q

What are the typical and abnormal charges on an amino acid?

A

Low pH (acidic): amino group protonated (–NH3+) and carboxyl group is not dissociated (–COOH)
Neutral: amino group protonated (–NH3+) and carboxyl group is deprotonated (–COO-)
High pH : amino group deprotonated (–NH2) and carboxyl group is deprotonated (–COO-)

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31
Q

What are different about the amino acid side chains?

A

They give distinct properties because they have unique size, shape, charge, H-bonding capacity, hydrophobic character and chemical reactivity.

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32
Q

What are the four groups of amino acid R groups?

A

Hydrophobic
Polar
Positively charged
Negatively charged

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33
Q

What are the hydrophobic R groups?

A

Alanine, Glycine, Phenylalanine, Proline Isoleucine, Leucine, Methionine, Tryptophan, Valine
(S attached to two C is non polar, indole ring is bulky and non-polar)
- Do not interact well with polar substances (ie water)
- Allows for compactness
-G: simplest
-W: bulkiest group with indole group casing conformity restriction

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34
Q

What are the polar R groups?

A

Asparagine, glutamine, cysteine, serine, threonine, tyrosine, histidine
- amide groups, thiol (sulfur bridges), alcohol, imidazole ring (H- reactive sights of enzymes, proton shuttle)

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35
Q

What are the positively charged R groups?

A

Arginine (guanidium group), Lysine

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36
Q

What are the negatively charged R groups?

A

Aspartic acid (aspartate)
Glutamic acid (Glutamate)
“ate” negatively charged carboxylic acid group

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37
Q

What is the primary structure of proteins?

A

The sequence of amino acids

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38
Q

What bonds amino acids?

A

amid bond aka peptide bond between O-C=O and NH3 loose H2O
Disulfide bonds: cross link by the oxidation of a pair of cysteine residues then called Cystine
*Oxidized - put together
*Reduced - go apart

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39
Q

What are the components of polypeptide chains?

A
  • Made of amino acid units called residues
  • Starts with amino-terminal residue ends with the carboxyl-terminal residue
  • Repeating: backbone or “main chain” repeating alpha Carbon, carbonyl, and amino group. Has H-bonding capacity C=O acceptor, NH donator
  • Variable: side chains/ R groups
  • naturally 50-2000 residues
  • small chains: oligopeptides (peptides)
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40
Q

What is the mean molecular mass of an amino acid?

A

100 g/mol
1 amu (chemists) = 1 Dalton (biologists)

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41
Q

What is the amino acid sequence important for?

A

Determining (guessing) the protein function, 3D structure, and reveals evolutionary history (how proteins change over time - antibiotic resistance)

aa alterations lead to abnormal protein function & disease

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42
Q

What is important to note about peptide bonds?

A
  • They are planar, 6 atoms— 2 alpha carbons, C=O and, NH are in the same plane
  • Resonance causes partial double-bond character prohibiting bond rotation
  • Most cases trans configurations are preferred (less steric hindrance) straightens chain (ie alpha carbons are on opposite side of bond)
    –Exception: proline because both confirmations have steric hindrance
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43
Q

What about peptide flexibility?

A

The torsional angels within an amino acid between the alpha carbon and its amino group (phi - knot or incredibles) or its carbonyl group (psi - trident) determines which confirmations are most likely/possible

non peptide bonds in the amino acid

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44
Q

What is a Ramachandran plot?

A

A plot of torsional angles (the different angels around the alpha carbon) to determine the most favorable angle
-White: too much hindrance to be possible
-Dark blue: most likely
represents the flexible nature of the non-peptide bonds

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45
Q

What is the secondary structure of a protein?

A

Formation of regular structures: alpha helices, beta pleated sheets, and turns

formed by H-bonds between N-H and C=O of amino acids near each other
(side chains determine preferrable angle but the h-bonding backbone holds it)

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46
Q

What are the components of alpha helices?

A
  • tightly coiled rod like structure
  • backbone works up and around as a helix bonded to itself every four residues (C=O w/ NH of fourth - 3 untouched residues in between)
  • R groups spread outward
  • almost all right handed
  • forms because energetically more favorable due to less steric clash between side chains
  • Drawn as ribbon or barrel
  • Amino acids that disrupt: V, T, I sometimes: S, N, D always: P
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47
Q

What are the components of B - pleated sheets?

A
  • Strands fold back on itself and H-bond
  • almost completely extended
  • R groups on amino acids alternate (one down the next up)
  • At least two B strands maybe more
  • Strands may be parallel (skewed bonding) or antiparallel (directly on top of each other)
  • May be more flat or become twisted
  • In a picture each sheet is one B strand interacting with the one next to it.
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48
Q

How are loops formed?

A

Reverse turn: when there is H-bonding with a residue three away.
Omega loop: no regular periodic structure but rigid and well defined

Enables polypeptide chain to change direction

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49
Q

What is an a-helical coiled coil?

A

right handed a-helixes coil L-handed giving the characteristics of Keratin (bonding allows wool to stretch and reform shape).

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50
Q

What is a collagen helix?

A
  • H-bonds absent within a strand.
  • Instead steric repulsion of pyrrolidine rings
  • 3 helical polypeptide chains. “superhelical cable”
  • G at every third residue b/c internally crowded
  • G-P-hydroxyproline common
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51
Q

What is the tertiary structure?

A

The overall shape of the whole entire polypeptide chain
- Globular or fibrous proteins (fully extended)

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52
Q

What is the characteristics of globular proteins and their tertiary structure?

A

highly compact, lack symmetry, soluble in water which is helpful for the aqueous environment in the cell
- Surface has charged amino acids with a compact interior of mostly nonpolar residues

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53
Q

What are the characteristics of the tertiary structure cell membrane proteins?

A

Since we do not want a highly charged protein in a fat layer
- outside: less polar/charged amino acids found here
- Inside: more polar/charged amino acids

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54
Q

What are protein motifs?

A

A combination of protein structures that show up in a lot of proteins (ie helix-turn-helix) these frequently exhibit the same function (ie common in DNA binding proteins)

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55
Q

What is a protein domain?

A

An independently folding region of a protein connected by short, flexible linker segments (ie CD4 with four domains)
- domains are determined by biochemists that may be using particular functions

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56
Q

What is the quaternary structure?

A

A unit composed of many different proteins
ie homodimer- two identical proteins, human hemoglobin - a2B2 tetramer, rhinovirus coat 60 copies of four subunits (proteins)
- Held together by numerous noncovalent bonds

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57
Q

How are disulfide bonds used to learn about protein folding?

A

Denature protein with urea and B-Mercaptoethanol disrupts disulfide bonds
When u and B-M are removed the sequence spontaneous reforms the original structure. Trace B-M is needed to ensure disulfide bonds are not scrambled.
The sequence determines the tertiary structure

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58
Q

What are the secondary structure preferences of E?

A

Glu, Glutamate, Glutamic acid

a-helix, reverse turn, B sheet

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59
Q

What are the secondary structure preferences of A?

A

Ala, alanine,
a-helix, Reverse turn/B sheet

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60
Q

What are the secondary structure preferences of L?

A

Leu, Leucine
a-helix/B sheet, reverse turn

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61
Q

What are the secondary structure preferences of M?

A

Met, Methionine
a helix, B sheet, reverse turn

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62
Q

What are the secondary structure preferences of Q?

A

Gln, Glutamine
a helix, B sheet, reverse turn

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63
Q

What are the secondary structure preferences of K?

A

Lys, Lysine
a helix, reverse turn, b sheet

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64
Q

What are the secondary structure preferences of R?

A

Arg, Arginine
a helix, b sheet/reverse turn

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65
Q

What are the secondary structure preferences of H?

A

His, Histidine
a helix, b sheet/reverse turn

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66
Q

What are the secondary structure preferences of V?

A

Val, Valine
b sheet, a helix, reverse turn

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67
Q

What are the secondary structure preferences of I?

A

Ile, Isoleucine
b sheet, a helix, reverse turn

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68
Q

What are the secondary structure preferences of Y?

A

Tyr, Tyrosine
b sheet, reverse turn/a helix

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69
Q

What are the secondary structure preferences of C?

A

Cys, Cysteine
b sheet, a helix, reverse turn

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70
Q

What are the secondary structure preferences of W?

A

Trp, Tryptophan
b sheet, a helix, reverse turn

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71
Q

What are the secondary structure preferences of F?

A

Phe, Phenylalanine
b sheet, a helix, reverse turn

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72
Q

What are the secondary structure preferences of T?

A

Thr, Threosine
b sheet, reverse turn, a helix

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73
Q

What are the secondary structure preferences of G?

A

Gly, Glycine
reverse turn, b sheet, a helix

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74
Q

What are the secondary structure preferences of N?

A

Asn, Asparagine
reverse turn, a helix, b sheet

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75
Q

What are the secondary structure preferences of P?

A

Pro, proline
reverse turn, a helix/b sheet

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76
Q

What are the secondary structure preferences of S?

A

Ser, Serine
reverse turn, b sheet, a helix

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77
Q

What are the secondary structure preferences of D?

A

Asp, Aspartic acid, Aspartate
reverse turn, a helix, b sheet

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78
Q

Can you predict the folding of a protein from its primary sequence?

A

No, one primary sequence section may fold differently in different proteins

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79
Q

What does it mean that proteins are a highly cooperative process?

A

Proteins tend to stick together or denature quickly only a very small amount will be 50/50. (intermediates do not accumulated)
- Because each step makes it want to go to the next step: Nuclear-condensation model
- Folding decreases entropy and increases enthalpy of surroundings (releases heat)
- Folded makes it the most happy, lowest enthalpy

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80
Q

How is the 3D folding of the protein predicted?

A

ab initio: from the beginning - computer based model take a sequence and calculate lowest entropy folding method (no extra protein knowledge)
Knowledge-based methods: rely on knowledge of known protein 3D structures

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81
Q

What are the important things to know about the methods of studying proteins?

A

We must isolate the protein and confirm that it was isolated.

We want it isolated so that we can learn the sequence of the protein

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82
Q

What is the method to purify a protein?

A

Get a cell, get a protein out of the cell
Start with an impure mixture then figure out what you want to isolate, isolate, the determine that you did isolate it

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83
Q

What is an assay and what does it do?

A

An assay is a test for a unique, identifying property of a protein.
A positive result of this test indicates that the protein is present
- Determines specific activity of a sample
- this can be done photometrically for enzymes by observing the absorbance of light of the intended product. (no absorb, no product, no protein)

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84
Q

What is specific activity?

A

It is the ratio of enzyme activity to the amount of protein in a mixture.
- The higher the specific activity the higher the purity of the mixture.

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85
Q

What are the actual steps to protein purification?

A

a) disrupt the cell membrane creating a homogenate
b) stick mixture in a centrifuge to separate molecular weights
c) purify with various methods including chromatography

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86
Q

What fractions does centrifuge create?

A

Nuclear then mitochondrial then microsomal fraction

continue centrifuging until you get the molecular weight of the protein you want to isolate

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87
Q

What is Levinthal’s paradox?

A

Protein folding cannot be at total random it would take too long only possible way would be if keep correct interactions

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88
Q

What are intrinsically unstructured proteins?

A

Proteins that completely or in part do not have a discrete 3D structure under physiological conditions

Regions are rich in charged & polar amino acids.

Assumes structure upon interaction–> ++ diff structiures and functions

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89
Q

What are metamorphic proteins?

A

Proteins with many structures of equal energy and when another molecule is added leads to different complexes

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90
Q

What amino acids prefer a-helices?

A

H, E, A, R, Q

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91
Q

What amino acids prefer b-sheets?

A

V, I, Y, C, W, T, F

92
Q

What amino acids prefer Reverse turns?

A

G, N, P, S, D

93
Q

What amino acids prefer both a helix and B sheets?

A

L, M

94
Q

What amino acid prefers a helix and reverse turns?

A

K

95
Q

What has been the effects of DNA sequencing?

A
  • Shown that genome sequences are similar between people (0.5% difference)
  • Variations in genome sequence can contribute to disease susceptibility
  • Genome sequence major role is to encode aa sequences of proteins
  • Show how long ago common ancestor
96
Q

How is protein versatility increased?

A

Posttranslational modifications
- chemical mod
- rearrangement of side chains
- Cleavage of peptide backbone

97
Q

What are the two relevant properties of water?

A

Polar molecule: bent, asymmetric distribution of charge, H: net positive, O: net negative
Highly cohesive: molecules interact strongly (ie ice)

98
Q

What comes from water being a polar molecule?

A

-High dielectric constant (measure of ability to store electrical energy)
-Interactions by hydrogen bonds
-Highly versatile because of interactions–readily dissolve many species (esp. polar & charged compounds)

99
Q

What are examples of Hydrogen-bond donors and acceptors?

A

Donor —– Acceptor
N-H —– :N
N-H —– :O
O-H —– :N
O-H —– :O

100
Q

What are the various forms of protein purification after centrifugation?

A

-Salting out:
-Dialysis
-Gel-filtration chromatography
-Ion-exchange chromatography
-Affinity Chromatography

101
Q

What is salting out?

A

adding salt to precipitate protein out of solution (as protein is attracted to salt it aggregates) protein becomes less soluble

102
Q

What is Dialysis?

A

Removal of small molecules from a protein using a semipermeable membrane that allows the small molecules out into solution purifying the protein within the membrane

103
Q

What is gel-filtration chromatography?

A

Separation of proteins by size
Pack column with beads of different size– something that will separate proteins
-smaller get farther down

104
Q

What is ion-exchange chromatrogrphy?

A

Separation of proteins by charge
Pack column with something to attract charge- negative/positive beads
Opposite charges attract then most alike charges pass through first

105
Q

What is Affinity chromatography?

A

highly specific,
specific molecule protein is attracted to–ligand–or if protein ligand unknown bind protein to polypeptide will bind to known ligand
all other proteins pass through

106
Q

What is HPLC?

A

High-performance liquid chromatography
Enhanced column technique: finer beads (> surface area) + pressure = sharper separations between proteins & > rapid separation
Measure protein absorbance of light create peaks – identifies presence of protein

107
Q

What is Gel Electrophoresis used for?

A

Telling that a purification scheme is effective by seeing how number of different proteins decrease with each purification step

108
Q

How does Gel Electrophoresis work?

A

Electrophoresis: molecule with net charge will move in electric field

Separated proteins by charge

109
Q

What happens in an SDS-PAGE?

A

Sodium dodecyl (12) sulfate adds negative charge and denatures proteins so the gel electrophoresis will work by mass only (SDS gives same charge to mass ratio)

PAGE: polyacrylamide gel electrophoresis

110
Q

What is isoelectric focusing?

A

Protein separation electrophoretically on the basis of contents of acidic and basic residues

It puts the proteins on a pH gradient. Proteins stop moving at their isoelectric point (pI) when they neutralize

Positive charge goes toward high pH side (-)
Negative charge goes toward low pH side (+)

111
Q

What is two-dimensional electrophoresis?

A

1st isoelectric focusing - that creates a horizontal pH separation based on protein charge
2nd SDS-PAGE to separate out by size

separates out similar proteins more

112
Q

If the purification is effective what changes should we see in the protein mix?

A

Total protein decreases, total activity decreases, specific activity increases, yield decreases, and purification level increases (current specific activity divided by initial specific activity)

113
Q

What is ultracentrifugation?

A

a method of higher velocity centrifugation for the analysis of physical properties of biomolecules

Measured in Svedberg units (S): small er S value moves more slowly.

The larger and more spherical the faster it moves into the end of the tube

114
Q

What does Recombinant DNA technology allow for?

A

Proteins can be expressed in large quantities (host organism, genetic manipulation)
Affinity tags can be fused to proteins
Proteins with modified primary structures can be readily generated (generate sections of protein)

115
Q

How is immunology used to investigate proteins?

A

Injecting a protein into an animal can produce antibodies that can be used to identify that protein (antigen)

116
Q

What is an epitope?

A

A specific group or cluster of amino acids on a target molecule aka antigenic determinant

117
Q

What are polyclonal antibodies?

A

Produced by multiple antibody-producing cell populations, a mix of proteins that recognize a different surface feature of the same antigen

118
Q

What are monoclonal antibodies?

A

clone of cells producing a single, identical antibody produces by immortal cell lines from multiple myeloma: hybridoma
Hybridoma cells can be screened by specific assay for the antigen-antibody interaction determine which produce antibodies of preferred specificity

119
Q

What can antibodies do for identifying proteins?

A

Can sensitively detect proteins when using ELISA and western blotting.
Labeling these antibodies can be used to visualize proteins within cells and tissues

120
Q

What is ELISA?

A

A standard test for protein-quantifying the amount of protein present (use beers plot)
When an antibody linked with an enzyme interacts with a substrate the color of the solution will change

121
Q

What is the difference between the two types of ELISA?

A

Indirect: Coat wall with antigen to identify, add antibody, add EL antibody, add substrate

Sandwich: Coat wall with specific monoclonal antibody, add antigen to identify, add EL monoclonal antibody, add substrate

122
Q

How does Western Blotting work?

A

SDS-PAGE to separate protein, transfer to polymer sheet, wash with primary antibody (specific for protein), add secondary antibody (specific for primary antibody), illuminate blot to measure florescence

123
Q

What is co-immunoprecipitation?

A

Another way to get a product out: antigen sticks to cell, add antibody to stick to antigen, and protein A bead that sticks to antibody the low speed spin brings beads to the bottom.

124
Q

How does mass spectrometry identify peptides and proteins?

A

Enables highly precise and sensitive measurement of the atomic composition of a particular molecule, analyte, without prior knowledge of its identity (don’t need to have a prior antibody)

Convert analyte to gas-phase ions to determine charge to mass ratio (m/z)

Two methods of conversion to gas-phase: Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization

125
Q

What is MALDI?

A

Matrix-assisted laser desorption/ionization hits the protein with a laser

126
Q

What is TOF?

A

Time of Flight determines mass by how long it takes for the protein to reach the detector

127
Q

What is the goal of MALDI TOF?

A

To determine the molecular mass of a protein

128
Q

What is Edman Degradation?

A

A method of taking off one amino acid at a time and using a mass spec to determine that changes in mass of the fragments.
Drawback: Limited to peptides of 50 residues
Solution: Chemical or Enzymatic cleavage with specific cleavage sites: more than one experiment run then piece fragments back together using overlap peptides

129
Q

What is tandem mass spectrometry?

A

The use of two mass spectroscopies one after another for more specific mass characterization.

Also called fingerprinting.

130
Q

What is the proteome?

A

Proteins expressed by the genome gives more information about types, functions, and interactions of proteins within its biological environment

131
Q

What can synthetic peptides be used for?

A
  • antigens to create specific antibodies
  • isolate hormone receptors and other signal molecules
  • study of nonproteinogenic amino acid effects on protein structure and function
  • drugs
  • define 3D protein structure rules.
132
Q

What can be used to determine the three dimentional structure of proteins?

A

X-ray crystallography and NMR

133
Q

What does x-ray crystallography do?

A

determine 3D position of most atoms within protein
- use x-rays to look at the scattering of protons causes diffraction that is interpreted as electron density by computers
- must first get protein into crystal (not easy)
- Better resolution: smaller angstroms, determined by crystal perfection
- Issue: not always in native form & proteins do not always crystalize

134
Q

How can NMR reveal protein structures?

A

One-dimensional: can resolve most protons in many proteins. Disturbs nuclei to create chemical shifts that can be associated with particular chemical group
Two-dimensional: gives more information from how spins of different protons affect their neighbors
- Nuclear Overhauser Enhancement SpectroscopY graphically displays pairs of protons in close proximity (even if not close in primary structure)

135
Q

What does cryo-electron microscopy do?

A

thin layer of protein solution frozen then put in TEM in vacuum and exposed to incident electron beam. Creates many projections assembles to 3D representation

136
Q

What are enzymes?

A

Biological catalyst: enhances the rate of a reaction without being consumed
(ie speed up half life many many times)
Most are proteins (ie hydration of CO2 in RBC requires carbonic anhydrase enzyme)
- exception: ie Ribosome - RNA catalyst

137
Q

What is an important characteristic of enzymes?

A

They have specific reactions and substrates
- some are more specific: one reaction or one substrate
- Some are less specific: one class of reactions, one class of substrate

138
Q

What do proteases do?

A

catalyze proteolysis, the hydrolysis (cleavage) of a peptide bond called a scissile bond
Creates: a carboxylate and a free ammoniate

139
Q

How are enzymes specific about cleavage?

A

Some cleave certain amino acid group peptide bonds (ie Thrombin & trypsin) some cleave almost any peptide bond (ie papain)

140
Q

How are enzymes named?

A

Names for substrate and for the reactions they catalyze ending with “-ase”

141
Q

What do enzymes cleave? How?

A

Ester bonds.
Peptide bonds: via hydrolysis

142
Q

What do enzymes do in reactions?

A

Catalyze both forward and reverse reactions to help establish equilibrium faster

143
Q

What are the six major classes of enzymes?

A

Oxidoreductases (oxidation-reduction), transferases (Group transfer), hydrolases (hydrolysis), lyases (addition/removal groups to form double bonds), isomerase (isomersation-intramolecular group transfer), ligases (ligation of two substates at the expense of ATP hydrolysis)

144
Q

What are co-factors? What are the terms that relate co-factors and enzymes?

A

Small molecules that are imperative for enzyme function (many enzyme activity depends on them)
- two classes: co-enzymes (vitamins/vitamin derivatives) and metals

  • Prosthetic: tightly bound coenzymes
  • Holoenzyme: enzyme has co-factor
  • Apoenzyme: enzyme without co-factor
145
Q

How do enzymes relate with energy?

A
  • Some generate ATP (photosynthesis, respiration)
  • Others use ATP to drive their action
    ATP is a co-factor to some enzymes
146
Q

How do enzymes relate to Gibbs Free energy and equilibrium?

A

Enzymes do not interact with Gibbs Free energy which only tells if the reaction is spontaneous or not and if equilibrium is reached (AG = 0, in equilibrium, AG > 0, endergonic reaction). Changing AG changes equilibrium (Keq) (just a 5kJ change AG can change equilibrium by a factor of 10)

Enzymes deal with rate not equilibrium

147
Q

What do enzymes facilitate?

A

Activation energy is necessary to get to the transition state. Decreasing the activation energy speeds the reaction

148
Q

What is AG*’?

A

Gibbs free energy under standard condition

149
Q

Where do enzyme reactions occur?

A

At the active site a specific region to stabilize the transition state more stable the more happy

150
Q

What is the first step in catalysis?

A

The formation of an enzyme-substrate complex in which substrates are bound at active-site clefts from which water is largely excluded

151
Q

What causes the specificity of enzyme-substrate interactions?

A

The arrangement of atoms in the active site (amino acids may be from different sections of the primary sequence)

Active site–small portion of the enzyme with a unique environment
The substrate is bound by weak attractions

152
Q

What is the evidence for the enzyme substrate complex?

A

x-ray crystallography high resolution of substrate and substrate analogs binding with the enzyme

153
Q

What is the enzyme substrate mechanism?

A

(Used to be thought: lock & key -perfect fit)
Now: induced fit as substate comes enzyme changes shape to produce the enzyme substrate complex

154
Q

What is binding energy? What is special about it and the transition state? What does it cause in enzymes?

A

The energy released on enzyme substrate binding
At the transition state is the tightest binding “full complete interaction” and the greatest amount of binding energy is released
Best stabilization
- Establishes substrate specificity and increases catalytic efficiency

155
Q

What happens if the transition state is too unstable or exists for too long?

A

It will collapse randomly to substrate or product
AG determines whether S or P accumulates

156
Q

What are the kinetic properties of enzyme reactions?

A

Simple reaction is A –> P with a rate of V determined by how much A disappears or how much P appears as a function of time

-First order reaction: V = k[A]
-Second order reaction: two reactants V=k[A]^2 or V=k[A][B]
*Most enzymatic reaction follows first order kinetics

157
Q

How is the initial rate of a reaction determined?

A

Since reactions are in equilibrium the only way to determine the true velocity is at t=0 when there is no product (P)
-Determined by determining the Vo at many different substrate concentrations then plotting it
-Must be experimentally determined multiple times
-Eventually getting to max velocity

158
Q

What is the Michaelis-Menton Kinetics? What is considered about the enzyme?

A

E+S <–k-1 –>k1 ES <–k-2 –>k2 E+P
At Vo there is no P so k-2 is 0

The enzyme is in a steady state, that is, the enzyme concentration does not change

159
Q

What is the use of Vo?

A

If Vo is plotted it will eventually reach near Vmax
1/2 Vmax is Km the Michaelis-Menton constant

160
Q

What is Km? What does it do? What is it equal to? What does a larger value mean?

A
  • The concentration of substrate at which the velocity is one half the maximal velocity
  • It tells us a lot about how an enzyme works (used in vivo)
  • Equals the equilibrium constant (Keq) first part of the Michaelis-Menton reaction
  • The larger the value the faster the enzyme works
161
Q

What is the Michaelis-Menton equation?

A

Vo = Vmax([S]/([S] + Km))

162
Q

What does it mean when the substrate concentration is equal to the Michaelis-Menton constant?

A

[S] = Km Vo = 1/2 Vmax

163
Q

What are the physiological consequences of Km?

A

When processing alcohol most people have a high and low Km. In conditions where people only have a high Km the reaction will happen quickly and they will have a worse hangover

164
Q

What is the Lineweaver-Burk Plot? What is the equation?

A

The double reciprocal plot of the Michaelis-Menton equation of 1/V vs 1/[S]
1/Vo = Km/Vmax X 1/[S] + 1/Vmax to produce a straight line
- y-intercept is 1/Vmax
- x-intercept is -1/Km
- slope is Km/Vmax

165
Q

What is kcat? What is its relationship to Vmax?

A

the turnover number: the number of substrate molecules converted to product per second
Vmax= kcat[E]t
The Vmax reveals the kcat if the [E]t (concentration of active sites is known)

166
Q

What things can the Km and Vmax can be used to determine?

A
  • Fes: the fraction of active sites filled
  • How the enzyme is behaving (–> why the enzyme works –> how to fix it)
167
Q

What classes do enzymes fall into?

A

Most enzymes in biological systems are bisubstrate either as a subsequential reaction or double-displacement

168
Q

What characterizes the subsequential reaction enzyme?

A

All substrates must bind to enzyme before the product is released. These can be ordered or random reactions, but no product will be released until all substrates are present.

169
Q

What characterizes the double-displacement reaction enzyme?

A

aka “ping pong reaction”
A substrate enters, reacts, and as a product leaves something behind (intermediate) then another substrate comes to pick it up
- almost none of these reactions progress without an enzyme

170
Q

What are allosteric enzymes?

A

Enzymes with positions that something binds to effecting enzyme activity but is not the active site
*Changes Michaelis-Menton plot to be sigmoidal

171
Q

How does temperature affect enzymes?

A

Increases in temperature increases enzyme rate

172
Q

What is the relationship between enzymes and observing molecules one at a time?

A

You can stick a molecule in with a lot of enzymes and see what happens. Reveals that enzymes at different folding states have different reaction properties. A change in reactivity shows enzyme flexibility.

173
Q

What are the two major types of enzyme inhibitors?

A

Irreversible enzyme inhibitors: bind covalently to enzyme and do not disassociate
Reversible inhibition: Capable of disassociation from enzyme. Creates enzyme-inhibitor complex noncovalently

174
Q

What are the types of reversible inhibitors?

A
  • Competitive inhibitors: blocks active site “competes for active site”
  • Uncompetitive inhibitors: bind to enzyme-substrate complex and stops the transformation (occurs after substrate binds)
  • Noncompetitive inhibitors: binds to enzyme allosteric site whether there is a substrate or not. Pure noncompetitive: doesn’t matter if the substrate is present. Mixed noncompetitive: has a preference if substrate is present or not.
175
Q

How are competitive inhibitors identified?

A

Since they create an Enzyme-inhibitor complex this effects the Km (it will need to be larger by adding more substrate to overcome equilibrium) and does not effect Vmax
x-intercept will be different (greater, less negative)
y-intercept will be unchanged

176
Q

How are uncompetitive inhibitors identified?

A

Since they create an enzyme-substrate-inhibitor complex it will not matter how much substrate is added the Km and the Vmax will be different (lower)
x-intercept will be different (smaller, more negative)
y-intercept will be different (greater)

177
Q

How are noncompetitive inhibitors identified?

A

Since it can create both an enzyme-inhibitor and an enzyme-substrate-inhibitor complex the Km is unchanged however the Vmax will be different
x-intercept will be unchanged
y-intercept will be different (greater)

178
Q

What are good competitive inhibitors?

A

Intermediate-analogs because they look very similar to the substrate

179
Q

What do irreversible inhibitors tell? What are the types?

A

Tell about the mechanism of action
- Group specific reagents: Covalently bond with a specific side chain
- Affinity labels: substrate analogs
- Mechanism-based (suicide) inhibitors: binds to active sight as a substrate but produces an intermediate that inactivates the enzyme

180
Q

What is the focus of enzymatic strategies? What are we really looking at?

A
  • What is going on in the enzyme to let the reaction take place
  • Binding energy: we want to decrease the binding energy by decreasing the activation energy with stabilizing weak interactions
181
Q

What causes induced fit?

A

Binding energy promotes a structural change in the enzyme and the substrate to facilitate catalysis

182
Q

What is the relationship between the enzyme and the binding energy?

A

Enzyme lowers binding energy (stabilizing the transition state) and destabilizes the intermediate so there will not be a dip in the energy diagram so the reaction can keep going.

183
Q

How do enzyme use strategies? What are the four strategies of enzymes?

A

They can use multiple strategies at once.
1) Covalent catalysis
2) General acid-base catalysis
3) Catalysis by approximation
4) Metal Ion Catalysis

184
Q

What is Covalent Catalysis? An example?

A

A temporary covalent bond between the enzyme and the substrate (ie proteases)

185
Q

What is General acid-base catalysis? An example?

A

Something other than water is the proton donator/acceptor important to get higher/lower pH than water (ie proteases and carbonic anhydrase, His is important)

186
Q

What is Catalysis by approximation? An example?

A

Brings two substrates together at a single binding site to enhance the reaction

187
Q

What is Metal ion catalysis? An example?

A

Any catalysis using a metal cofactor- often Mg or Ni
(ie carbonic anhydrase)
- May facilitate nucleophile, be an electrophile or a bridge between the enzyme and substrate

188
Q

What are the thermogenetic and kinetic properties of proteases?

A

It is thermogenetically favorable (AG= -)
Activation energy is very high because of resonance that makes the carbonyl carbon less reactive.

189
Q

What is the specificity of chymotrypsin?

A

It wants to cleave large hydrophobic proteins.

190
Q

What is a very important part of chymotrypsin?

A

Serine because it creates a covalent bond using its alcohol group (Covalent catalysis)

191
Q

How do we know the importance of serine to chymotrypsin?

A

When DIPF experiment modified the Serine in the active sight the enzyme became inactive. (Suicide inhibition)
Also Phosphorus-Based Agents (Malathion and Sarin) both inactivate protease by modifying serine.

192
Q

How was the reaction of chymotrypsin measured and the strategy determined?

A

N-acetyl-L-phenylalanine-p-nitrophenyl ester because proteases destroy esters creating a color change

Determined this was a covalent catalysis because at first there was a burst of color (acylation) and then there was a slower second step (Rate determining Step) (deacylation - add water to cleave and free enzyme)

193
Q

What are the three aspects of the chymotrypsin enzyme reaction?

A

Reaction at the active sight with a catalytic triad
Stabilization of the negatively charged oxygen with an oxyanion hole
Selectivity of amino acids via a binding pocket

194
Q

What is the catalytic triad of chymotrypsin and how does it work?

A

Serine, Histidine, and aspartic acid to make the serine alcohol more nucleophilic
Asp carboxylate takes a proton/hydrogen bonds with His that acts as a proton shuttle to take a proton from Ser. Ser is then a good nucleophile to attack the substrate carbonyl carbon making it a, sp3, tetrahedral intermediate and giving the oxygen a negative charge. Then the scissile bond breaks and the amine takes the proton from His and leaves. (Acyl-enzyme, fast)
Then H2O is added and one proton goes to His and the OH attaches to the carboxylic acid component (tetrahedral intermediate) then the carboxylic acid component leaves and the Ser takes a proton from the His.

195
Q

What makes proteases specific? Examples?

A

The side chain binding position and binding pockets.
- Chymotrypsin: long deep S pocket for large hydrophobic group (hydrophobic because pocket is carbon based)
- Trypsin: Asp in binding pocket for positive groups
- Elastase: 2 Val for small hydrophobic groups

196
Q

What is S1, S’1, P1, P’1 etc?

A

The numbering system for sidechains and binding pockets
S is for binding pocket
P is for sidechain
No ‘ is for the amino-terminal side of the scissile bond (C- scissile)
‘ is for the carbonyl-terminal side of the scissile bond (scissile-N)
The number indicates the residue from the scissile bond

197
Q

What does the oxyanion hole do?

A

Stabilizes negative charges either with H-bond donors or with electrostatic (positively charged0

198
Q

What did mutation tests reveal about proteases?

A

Mutation decreased the activity (Kcat) significantly but not slower than noncatalyzed reactions (mutated catalysts still brought reactants closer together, Proximity)
And did not change Km indicating that the Enzyme-substrate complex was still made

199
Q

What are some examples of proteases using things other than serine?

A
  • Cystine proteases: Uses thiol group
  • Aspartyl Proteases: use carboxylate (a pair of Asp)
  • Metalloprotease: Activates H2O by deprotonating a base to attack the carbonyl
    *Most still have oxyanion to stabilize negative oxygen
200
Q

What do proteases do in common?

A
  • Activate H2O or other nucleophile
  • Polarize peptide in a carbonyl group
  • Stabilize tetrahedral transition state
201
Q

What do important protease inhibitors do?

A

Blood pressure drugs inhibit ACE protease (metalloprotease)
AIDS drugs inhibit the protease of HIV that activates a virus

202
Q

What is the function of carbonic anhydrase?

A

It catalyzes t
he conversion of water and carbon dioxide into carbonic acid (byproduct of cellular respiration that we do not want in the blood)
There are many different carbonic anhydrases but they are all very similar
Rate max at pH = 8, midpoint at pH of 7

203
Q

What are the major aspects of carbonic anhydrases? (what are the catalytic strategies)

A

Zn 2+ bound to 4 ligands (3 His imidazole rings, H2O): Zinc-water complex
Catalytic strategies: Metal ion, approximation (H2O and CO2), and General Acid-Base

204
Q

What does Zinc do for carbonic anhydrase?

A
  • Creates a Zinc Water complex
  • Zinc lowers the pKa of H2O (15.7) to 7 (easily deprotonated) to increase the likelihood of forming a hydroxide ion.
  • Zinc also stabilizes the negatively charged oxygen ion when OH reacts with CO2
205
Q

What does the carbonic anhydrase zinc water complex do?

A

A hydrophobic path in the enzyme adjacent to the Zn-HSO complex brings CO2 in close proximity to the Zn-H2O complex
- OH creates a carbonyl group with CO2 and the O- is stabilized by the Zinc
- OH- reacts with CO2 to create HCO3-

206
Q

Even though the H2O is easily deprotonated, it is not easily enough for the reaction rate observed. What does carbonic anhydrase also have to make the reaction faster than its normal equilibrium constant?

A

(something to pull the proton off)
Another His works as a proton shuttle taking one proton with a N and shuttling it to the surface of the molecule to an awaiting buffer as the other N swings around to take another proton.
This is in equilibrium so the process can go in both ways

207
Q

What is the mechanism of carbonic anhydrase?

A

Zn-H2O complex looses a proton (proton shuttle with His), now Zn-HO-, CO2 is added, O- attacks C making one of the CO2 O’s negatively charged, Zn stabilizes the negatively charged O, another H2O comes and the HCO3- leaves

208
Q

What do restriction endonucleases/enzymes do? What are we focusing on?

A

Destroy foreign DNA while leaving Host DNA alone
Focus on Type II Restriction Enzymes that cleave DNA at recognition sequence

209
Q

How does Type II Restriction Enzymes work? (cleavage site, result, attack)

A

They cleave the bond between the 3’-oxygen atom and the phosphorus atom resulting in a free 3’-hydroxyl group and a 5’-phosphoryl group at the cleavage sight
By a nucleophilic attack at the phosphorus atom

210
Q

What are the two mechanisms of Type II Restriction Enzymes?

A

1) Covalent intermediate employing a potent nucleophile, a double displacement reaction (retains stereochemistry)
2) Direct hydrolysis, single displacement inverting stereochemistry

211
Q

What does in-line displacement mean?

A

Nucleophile attack phosphorus atom forming a pentacoordinate transition state.

212
Q

What is the problem for determining between the two mechanisms of Type II Restriction Enzymes?

A

There are two oxygen atoms so the molecule is not technically chiral.
Solution: replace an O and use labled water.
Result: there was inverted stereochem so this was by the second mechanism

213
Q

How do Type II Restriction Enzymes have special selectivity of the sequence?

A

the recognition sequences are inverted repeats and the restriction enzymes have a corresponding symmetry: dimers (two subunits related by twofold rotational symmetry)

214
Q

What do many enzymes that act on phosphate-containing substrates require?

A

Mg2+ or some other similar divalent cation for activity.

215
Q

What does Mg2+ do for Type II Restriction Enzymes?

A

Stabilized by two Asp binds with H2O to position and activate H2O.
Asp is in a position to hydrogen bond to H2O so the Oxygen can attack the Phosphate causing cleavage

216
Q

What is special about the recognition sequence of of most type II Restriction Enzymes?

A

They are inverted repeats if you rotate them 180* they are still the same. 5’ - GATATC - 3’ 3’-CTATAG-5’
Twofold rotational symmetry
- The enzyme have a corresponding symmetry: dimers with two subunits
- a kink in the DNA allows it to get close enough to the active site to react

217
Q

Why does EcoRV only cleave cognate sequences when it binds with equal affinity to all sequences?

A

There is distortion of the DNA: there is a kink in the center created by the TA base pairs in the recognition sequence. Puts phosphate close enough to active site to react (increases binding energy)

218
Q

Why do restriction endonucleases not destroy host DNA?

A

DNA methylase adds a methane to Adenine bases in recognition sequences preventing the adenine from getting close enough to the enzyme to react.

219
Q

What are myosins? What do they do?

A

Molecular motor proteins (actually move)
- Walk across actin
- Catalyze the hydrolysis of ATP to ADP and Pi a thermodynamically favorable reaction

220
Q

What is the structure of myosin?

A

Elongated dimer structure that wrap around each other with globular domains (ATPase) that carry out ATP hydrolysis

221
Q

What are the aspects of myosin ATPase domains?

A
  • Almost always have Mg2+
  • Not much conformational change even with both ATP and Mg2+ present until reaction occurs
  • Mg2+ to far away from Phosphate group to react so there must be a conformational change
  • Mn2+ can also work
    Other: NTP (nucleotide) requires Mg2+/Mn2+ (otherwise inactive)
  • Metal ion is a substrate not an aspect of the active site
222
Q

What does the active site of the myosin ATPase contain?

A
  • Transition state is pentacoordinate as the H2O attacks the Phosphate (confirmed by Vanadium replacement)
  • Ser is important as the ATP takes its H and Ser takes H2O H so the OH can attack the phosphate
223
Q

What does the myosin ATPase reaction result in?

A

Creates a little swing that it amplified to a big step by the myosin’s long arm

224
Q

What is the rate of the myosin ATPase reaction? Why?

A

It is a slow enzyme with a turnover of about once per second
- The RDS is the detachment of Pi (the release of products) – critical for the coupling of conformational changes
- discovered by using 18O in H2O found that 2-3 of the oxygen atoms in the phosphate were derived from water indicating that the ATP hydrolysis reaction within the enzyme active site is reversible. (Gets O from H2O and cleave rotates, reforms many times before product released)

225
Q

What is the mechanism of myosin movement along an actin filament?

A

1) ATP binding detaches Myosin from Actin
2) (+ H2O) the reversible hydrolysis of ATP bound to M can reorientate lever arm
3) ATP hydrolyzed: ADP-Pi and sill bound to M can still bind to A
4) Release of Pi reorientates the lever arm (other foot moves)
5) Release of ADP from M completes cycle.

CU: Biochemistry (4 decks)

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