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Bioc 2580 > Midterm Study Set > Flashcards

Midterm Study Set Flashcards

1
Q

What is the protein that stores oxygen in muscle tissue

A

myoglobin

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2
Q

What measurement is protein size measured in

A

kilodaltons

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3
Q

What are the building blocks of proteins

A

amino acids

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4
Q

What bond holds amino acids together

A

peptide bonds

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5
Q

What is the positive end of an amino acid structure

A

amino group

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6
Q

What is the negative end of an amino acid structure

A

carboxylate group

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7
Q

What differentiates the 20 amino acids

A

the R groups

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8
Q

What are the 2 possible spatial arrangements of amino acids

A

L and D

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9
Q

For stereoisomers, what is the term for mirror images (D and L configurations)

A

enantiomers

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10
Q

What configuration are proteins found in

A

L configuration: amine group of the left (L for left)

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11
Q

What is a condensation reaction

A

cleavage of H2O

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12
Q

What is a hydrolysis reaction

A

using H2O to form a bond

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13
Q

Where is the point of weakness on a peptide bond where H2O can form/break bonds

A

the C=O bond

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14
Q

In which direction does the equilibrium of the reaction lie under standard biochemical conditions (in terms of hydrolysis and condensation)

A

hydrolysis

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15
Q

In order for the reaction of a peptide to move in the direction of peptide bond synthesis, what must occur

A

carboxyl group needs to be chemically modified

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16
Q

How are polypeptides and oligopeptides different

A

polypeptides = many amino acids
oligopeptides = a few amino acids

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17
Q

Why are the amino acids in a chain known as amino acid residues

A

because when water is cleaved off during peptide bond synthesis, some molecular weight of the amino acid is also cleaved, leaving behind just the amino acid residue in the chain

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18
Q

How can the number of amino acid residues be estimated given the molecular weight of a protein

A

divide by 110
(the molecular weight of an amino acid is ~128, and the weight of water cleaved off is ~18, therefore dividing by 110 (the weight of one residue) gives an estimate to the number of amino acids present

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19
Q

What carbon is the central carbon in the backbone (think in terms of greek letters)

A

alpha carbon

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20
Q

What is the order of carbons in an amnio acid chain (think in terms of greek letters)

A

alpha, beta, gamma, delta, epsilon

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21
Q

What are the 3 properties to consider between the different amino acid side chains

A

polarity, charge, and H-bond ability

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22
Q

What are the 6 very non polar amino acids

A

Alanine, Valine, Leucine, Isoleucine, Methionine, and Phenylalanine

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23
Q

What are the 5 non polar amino acids

A

Glycine, Cysteine, Proline, Tyrosine, and Tryptophan

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24
Q

What are the 4 uncharged but polar amino acids

A

Serine, Threonine, Asparagine, and Glutamine

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25
Q

What are the 3 positively charged amino acids

A

Lysine, Histidine, and Arginine

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26
Q

What are the 2 negatively charged amino acids

A

Aspartate and Glutamate

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27
Q

If an amino acid is non-polar is it hydrophilic or hydrophobic

A

hydrophobic

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28
Q

If an amino acid is polar is it hydrophilic or hydrophobic

A

hydrophilic

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29
Q

Why do polar molecules interact with water

A

polar attracts polar, and water is a polar molecule as well

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30
Q

What is the sequence of electronegativity of elements found in amino acid chains

A

O>N>S>C=H (O being most electronegative, C and H being least)

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31
Q

Why would an atom with a longer CH chain be considered more non-polar

A

C=H bonds are non polar, but the more interactions present the more non-polar the molecule is

ie. Glycine is only moderately non polar because its side chain consists of one single hydrogen, not many

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32
Q

What about Serine and Threonine make them polar but still uncharged

A

the hydroxyl group on the R chain

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33
Q

What about Aspirigine and Glutamine make them polar but still uncharged

A

the amide group on the R chain

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34
Q

What are good examples of hydrogen donors for H-bonds

A

OH- or NH- groups

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35
Q

What are good examples of hydrogen acceptors

A

O or N atoms with lone pair of electrons

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36
Q

What about Lysine, Histidine, and Arginine make them positively charged and polar

A

the NH3+/NH2+/NH+ groups on the R chain

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37
Q

What about Aspartate and Glutamate make them negatively charged

A

deprotonated R chains with a carboxylate group (COO-)

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38
Q

What are the 7 AAs that can donate/accept a proton on its R chain

A

Aspartate, Glutamate, Tyrosine, Cysteine, Arginine, Histidine, Lysine

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39
Q

What are the 4 AAs that go from neutral to negatively charged when deprotonated

A

Aspartate, Glutamate, Tyrosine, and Cysteine

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40
Q

What are the 3 AAs that go from positive to neutral when deprotonated

A

Lysine, Arginine, and Histidine

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41
Q

Henderson-Hasselbalch equation

A

pH = pKa + log(deprotonated/protonated)

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42
Q

Why are amino acids considered dipolar

A

has both a +ve and -ve charge (NH3+ and COO-)

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43
Q

What happens when pKa=pH

A

50% of AA is in protonated form and 50% is deprotonated

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44
Q

Why does deprotonation occur with raised pH

A

[H+] becomes less available so deprotonation is more likely to occur

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45
Q

If the pH is one or more units below the pKa, the AA is…

A

protonated

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46
Q

If the pH is one or more units above the pKa, the AA is…

A

deprotonated

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47
Q

If the pH is less than one unit above or below the pKa then what happens…

A

calculation is needed

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48
Q

What is amino acid analysis and what two processes does it involve

A

functions that help determine protein structure
- involves separation and detection

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49
Q

What is partition chromatography (column chromatography)

A

stationary phase resides in a column and a mobile phase is run through the column to separate amino acid proteins

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50
Q

How are amino acids measured from column chromatography

A

concentration of protein(s) is measured in the elution volume

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51
Q

What is thin layer chromatography

A

silica gel is spread across a thin sheet of plastic and samples are applied near the lower edge and placed in solvent, proteins then soak upwards in the gel based on polarity

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52
Q

Which proteins travel further in gel filtration

A

non-polar amino acids

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53
Q

What is the stationary and mobile phases of gel filtration

A

stationary = plastic sheet
mobile = solvent buffer

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54
Q

What is added to gel filtration that indicates proteins present

A

ninhydrin

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55
Q

Ion chromatography separates based on __________

A

charge

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56
Q

How does a cation exchange resin work

A

contain negative groups within the resin, so bind to positive groups in sample

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57
Q

How does an anion exchange resin work

A

contain positive groups within the resin, so bind to negative groups in sample

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58
Q

What determines how tight proteins bind to an ion exchange resin

A

the strength of the charge (more positive/negative will bind tighter than less positive/negative, regardless of the fact that they are both positive/negative)

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59
Q

Where are amino acid concentrations measured in ion exchange chromatography

A

the elution volume

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60
Q

What is affinity chromatography

A

ligand is covalently attached to the beads in the resin and proteins that have an affinity to the ligand bind tightly to it while others pass through the column
- proteins bound to the ligand are then removed with the addition of high-concentration salt or other ligand

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61
Q

What is a “tag” in relation to a ligand

A

a peptide/protein that is fused to the target protein and is capable of binding to a ligand

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62
Q

What is metal affinity chromatography

A

column has a resin containing metal ions that the tagged proteins can bind to
- adding a competitor to the tagged protein out competes the tag and allows those proteins to elude

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63
Q

What is a benefit of metal affinity chromatography

A

high purification in minimal steps

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64
Q

What characteristic does gel filtration chromatography seperate proteins based on

A

allows separation of proteins based on size

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65
Q

How does gel filtration chromatography work

A

polymeric gel resin that consists of many water-filled pores, large molecules don’t fit in the pores but small do, so large elude first then small later on

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66
Q

What is gel electrophoresis

A

sample is injected into a gel and the proteins move along the gel based on charge

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67
Q

What is SDS PAGE

A

sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)

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68
Q

How does SDS PAGE work

A

proteins are treated with SDS, so all have same uniform charge, separation is based solely on size
- smaller migrate faster than large

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69
Q

What is isoelectric focusing

A

separation based on the isoelectric point of proteins
- when net charge of the protein is 0, the pH at which it lies is its isoelectric point

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70
Q

What are two dimensional gels

A

combine SDS PAGE and isoelectric focusing

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71
Q

What is mass spectrometry

A

protein is vaporized by a laser beam and particles travel toward the detector
- velocity at which the particles travel depends on mass

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72
Q

How to determine the protein in sample in mass spectrometry

A

using a database of protein masses

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73
Q

What is an enzyme unit

A

the amount of enzyme that converts one umol of substrate to product per min

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74
Q

What is enzyme activity

A

total units of enzyme present in a solution

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75
Q

What is the specific activity of an enzyme

A

number of enzyme units per milligram of total protein

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76
Q

What is the equation for specific activity

A

specific activity = (enzyme activity/total protein)

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77
Q

With purification, does specific activity increase or decrease

A

specific activity increases with each purification step (based on the equation you’d be dividing by smaller values)

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78
Q

When samples of pure and unpure versions of the SAME enzyme are examined, what can be determined

A

enzyme purity

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79
Q

With purification of a protein, what happens to enzyme activity

A

decreases

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80
Q

With purification of a protein, what happens to total protein

A

decreases

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81
Q

With purification of a protein, what happens to total protein

A

increases

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82
Q

How are amino acids separated from a chain to begin with

A

hydrolysis of peptide bonds

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83
Q

What is a nucleophile

A

an atom with a lone pair of electrons available to share

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84
Q

What is an electrophile

A

a lone pair seeking atom (wants an atom to come bond with it)

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85
Q

How can an atom with a lone pair use it

A
  1. by h-bonding
  2. as a base (captures [H+])
  3. as a nucleophile
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86
Q

What is nucleophilic substitution

A

incoming nucleophile attacks the target atom to displace the leaving group (leaving group takes bonding electrons with it)

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87
Q

What is nucleophilic addition

A

in cases where the target atom is double-bonded to the leaving group, only one bond has to be given up to the leaving group does not disconnect

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88
Q

Are carbon-carbon bonds good or bad leaving groups

A

bad; C-C bonds are hard to break so they are poor leaving groups

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89
Q

How many helical segments are in myoglobin

A

8

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90
Q

What is primary structure in a protein

A

the linear sequence of amino acids

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91
Q

What is secondary structure of a protein

A

the repetitive patterns of the peptide chain (ie. helices or pleated sheets)

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92
Q

What is the tertiary structure of a protein

A

the overall 3D pattern of a single polypeptide

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93
Q

What is quaternary structure of a protein

A

combining multiple polypeptides to form a protein complex

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94
Q

What scientist worked with insulin and developed the method of investigating proteins using fluorodinitrobenzene

A

Fred Sanger

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95
Q

How does fluorodinitrobenzene work to investigate proteins

A

the free amino group on the amino acid chain deprotonates and acts as a nucleophile and seeks out the fluorodinitrobenzene reagent
- HF on the reagent acts as a leaving group and the reagent binds
- hydrolysis releases the N-terminal amino acid with the yellow tag attached to be identified by chromatography

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96
Q

What is the downfall to fluorodinitrobenzene as an investigative method of protein

A

can only investigate the first amino acid of the sequence (hydrolysis destroys the rest of the chain)

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97
Q

Who improved the Sanger method

A

Perh Edman (Edman degradation)

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98
Q

What is the main difference between the Sanger method and Edman degradation

A

Edman degradation can be repeated and is done without hydrolyzing the rest of the chain

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99
Q

What are the two steps of Edman degradation

A

coupling and cyclization

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100
Q

What does coupling require in Edman degradation

A

base

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101
Q

What does cycling require in Edman degradation

A

acid

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102
Q

How many times can the Edman degradation cycle be repeated

A

up to 50 times

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103
Q

What is the purpose of selective hydrolysis

A

to cut long protein chains into smaller oligopeptides (divide and conquer type method)

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104
Q

What does the digestive enzyme trypsin bind and recognize

A

Arg and Lys

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105
Q

What is the exception to the binding and cutting of Arg and Lys

A

cannot occur when proline is the following AA

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106
Q

What is the positioning of Arg/Lys to trypsin when the protein is cut

A

the carboxylate end of Arg or Lys is positioned at the catalytic site of trypsin

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107
Q

WHY does hydrolysis of Arg or Lys not work when followed by proline

A

proline doesn’t fit correctly into the catalytic site which prevents the ability to cut

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108
Q

What does chymotrypsin act on

A

tyrosine, tryptophan, and phenylalanine

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109
Q

WHY does the hydrolysis of Tyr, Trp, and Phe not work when followed by proline

A

similarly to the previously stated with trypsin, the proline AA does not fit into the catalytic site of chymotrypsin either

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110
Q

What does cyanogen bromide act on

A

chemical reagent that acts on methionine residues

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111
Q

How does cyanogen bromide work

A

acts on methionine residues and turns methionine into homoserine (Hse)

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112
Q

What is the overlap method

A

two samples of the same original oligopeptide are cut using trypsin and chymotrypsin (targeting the different corresponding sites) and the strands from each set are lined up to overlap one another and determine the overall original sequence

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113
Q

How are most amino acid sequences derived now

A

using DNA sequences to then predict the original amino acid sequence

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114
Q

What is tandem mass spectrometry

A

2 mass spectrometers work together in tandem
- first MS-1 sorts different peptides and select one type to go into collision cell
- fragments each peptide in a random fashion
- second MS-2 measure the fragment masses

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115
Q

What is the protease of choice in mass spectrometry

A

trypsin

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116
Q

How does trypsin play importance to mass spectrometry

A

trypsin is the protease of choice: breakage in the collision cell results in b-type (N terminal) and y-type (C terminal) fragments; all y-type will have Arg or Lys on the c-terminal since trypsin is used, and mass spectrometry produces signals based on charge

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117
Q

How to find mass of amino acid using data from mass spectrometry

A

difference in mass from largest peak to next largest peak gives mass of unknown AA (calculate backwards)

118
Q

What is BLAST searching

A

basic local alignment search tool
- compares input sequence to a database to confirm protein sequences or discover new ones

119
Q

How is the backbone of a polypeptide chain flexible

A

bond rotation

120
Q

How does bond rotation work

A

groups connected by single bonds can rotate about a bond axis

121
Q

Do bonds bend?

A

NO

122
Q

What are conformations

A

represent states of a molecule that can be interconverted by bond rotations without breaking covalent bonds

123
Q

What are configurations

A

can only be interchanged by breaking covalent bonds, not by rotation

124
Q

What are examples of configurations of molecules

A

cis and trans

125
Q

What are the two chiral configurations of amino acids

A

L and D

126
Q

What does X-ray diffraction measure

A

regular repeating patterns on the molecular scale
- dimensions of the repeating pattern can be calculated

127
Q

Who interpreted the fibre patterns in fibrous proteins

A

Linus Pauling

128
Q

What was Paulings key finding

A

peptide bond has double bond character

129
Q

How did Pauling determine his conclusions

A

length of a peptide bond was closer to that of a double bond than a single bond, therefore it has more double bond character

130
Q

Is a peptide bond flexible or rigid

A

rigid and fixed

131
Q

Why is a peptide bond rigid

A

because it has double bond character

132
Q

Within restricted bond rotation, what are the possibilities for structure and how are they arranged

A

helical: a-c bond down peptide chain turns in same direction
extended: a-c bond turns in alternate direction
no shape: random coil

133
Q

How many amino acids occur per turn of the alpha helix

A

3.6 amino acids

134
Q

How many amino acids are required minimum to create an alpha helix

A

5

135
Q

What is the distance between each turn of the alpha helix

A

5.4 Armstrongs

136
Q

What is the distance along the alpha helix per amino acid

A

1.5 Armstrongs

137
Q

Where is the hydrogen bond found in the alpha helix that holds its shape together

A
  1. C=O lines up with 5. H-N
    (2. C=O lines up with 6. H-N, etc.)
138
Q

In relation to beta sheets, strands in opposing directions form _______________ B-sheet, whereas strands in same direction form _________________ B-sheet

A

antiparallel
parellel

139
Q

Which amino acids prefer beta pleated sheet orientation

A

Trp, Tyr, Phe (all big in size), Val, Ile, Thr (have a branch on B-C), and Cys (large S atom on B-C)

140
Q

What determines the overall shape of an AA strand

A

local majority - does the majority prefer alpha helix or beta sheet orientation

141
Q

What are the 5 breakers of secondary structure

A

GPNDS (glycine, proline, arginine, aspirigine, and serine

142
Q

How many breakers are required in what amount of space to effectively work

A

2 breakers in a group of 4 amino acids

143
Q

What is the point of a breaker

A

forms a flexible loop or turn and allows the polypeptide chain to change direction drastically

144
Q

What is denaturation

A

when a protein unfolds as a result of unnatural environmental factors

145
Q

What are ways to denature a protein

A

heat, disruptive solvents, SDS

146
Q

What is the simplest form of tertiary structure of polypeptide

A

no breakers; no rotation

147
Q

What has a unique triple helix structure in our bodies

A

collagen

148
Q

Since the structure of proteins is rigid, they are fibrous, what does this mean?

A

not able to fold

149
Q

If a-keratin is fibrous, what does this mean about its structure

A

it has no breakers

150
Q

What amino acids are enclosed towards the middle

A

non-polar

151
Q

What amino acids are on the exterior of the folding

A

polar

152
Q

Why are non-polar amino acids on the interior of folding pattern

A

because of the hydrophobic effect, want to minimize contact with H2O

153
Q

Why are polar amino acids found on the exterior of folding pattern

A

interact well with H2O (good H-bonding)

154
Q

What forces are at play when proteins fold together in a jigsaw puzzle effect (tightly interlocked)

A

weak van der Waal forces by close contact

155
Q

With a sequence of mostly a-helices, what overall structure will form

A

a-helix bundle

156
Q

What creates a non-polar patch/stripe

A

non-polar amino acids every 3 or 4 places in an AA sequence that fold inside the bundle

157
Q

What is more stable; antiparallel or parallel beta sheets

A

antiparallel: H-bonds are arranged in a straight line (diagonal in parallel)

158
Q

When a sheet is polar on one side and non-polar on the other side, what happens

A

the sheet folds with non-polar facing inwards to form a beta barrel

159
Q

Sequences with alternating alpha and beta configuration can form what

A

a parallel beta sheet

160
Q

Where do the helices lie on a parallel sheet

A

above or below the plane of the sheet

161
Q

Why are parallel pleated sheets less stable

A

the H-bonds run on a diagonal

162
Q

If parallel pleated sheets are usually buried in the centre of a protein, what kind of amino acids is it typically made of

A

non-polar

163
Q

If all helices lie on the same side of a parallel sheet, what configuration does it take on

A

alpha beta barrel

164
Q

What is an alpha beta barrel

A

a parallel sheet where all helices lie on the same side of the sheet, so it folds into a central barrel surrounded by connecting a-helices

165
Q

If the helices on a parallel sheet lie on both sides of the sheet, what configuration does it take on

A

alpha beta sandwich

166
Q

What is an alpha beta sandwich

A

a parallel beta-sheet where alpha-helices lie on both sides of the sheet (think sheet is sandwiched by helices)

167
Q

What are domains in respect to large proteins

A

larger proteins fold up into sections called domains (ie. a protein might have 2 ab sandwich domains)

168
Q

What is the native state of a protein

A

its normal, folded state

169
Q

What is the term for when a protein folds and loses function

A

denaturation

170
Q

Where is covalent bonding found in proteins

A

covalent bonding links amino acids (peptide bonds are covalent)

171
Q

Where us non-covalent bonding found in proteins

A

dictate the folding pattern and stability
(most importantly the hydrophobic effect and van der Waals forces)

172
Q

What are the most important non-covalent bonding features of proteins

A

hydrophobic effect and van der Waals forces

173
Q

How much total energy of native state does the hydrophobic effect contribute

A

50%

174
Q

What is another term for van der waals forces

A

London dispersion forces

175
Q

What are van der Waals forces

A

weak electrostatic forces between atoms that are close in proximity

176
Q

What is the important note regarding van der Waals forces in proteins

A

need many atoms in close contact to actually have an effect, since they are weak interactions

177
Q

How is a salt bridge formed

A

negative side chains pair up to positive side chains nearby and create a salt bridge in the folded protein

178
Q

Do ion pairs and H-bonding contribute more or less to tertiary protein folding compared to hydrophobic or van der Waals interactions

A

less impact, van der Waals and hydrophobic interactions have a greater effect

179
Q

Why do van der waal forces and the hydrophobic effect have a greater impact on protein folding

A

because polar AAs face the exterior and these forces target polar AAs

180
Q

What forces have greatest effect on secondary protein folding

A

H-bonds

181
Q

What kind of bonds help hold tertiary structure of proteins

A

disulphide bonds

182
Q

How do disulphide bonds form in tertiary protein structure

A

pairs of SH groups (from cysteine AAs) react with O2 and release H2O

183
Q

Are disulphide bonds common in proteins

A

no; only few have them and those are likely proteins that function outside cells (since O2 is needed)

184
Q

What is the experiment done by Christian Anfinsen

A

urea weakens the hydrophobic effect and unfolds protein
2-mercaptoethanol acts as a reducing agent that converts disulphides back to original unlinked Cys-SH groups (reduces disulphide bonds)
once urea and 2-mercaptoethanol were removed the protein renatured, proving the amino acid sequence contains all the info needed for folding

185
Q

Non polar patches on proteins bind by what effect

A

hydrophobic effect

186
Q

Matching shapes on proteins bind by what effect

A

van der Waals forces (maximized close contact)

187
Q

Match of charged groups bind by what effect

A

H-bond effect (or just charges)

188
Q

What do enzymes recognize and bind

A

enzymes recognize and bind specific target proteins and catalyze reactions

189
Q

What do antibodies recognize and bind

A

bind and identify foreign molecules in the body

190
Q

What amino acids fit best in the chymotrypsin binding pocket

A

Phe, Tyr, and Trp

191
Q

How does chymotrypsin bind to polypeptides to find Phe, Tyr, and Trp

A

groove in chymotrypsin allows AA chain to pass through (think a chain passing through a tubular hole in an enzyme)
binding pocket is large and surrounded with non-polar AAs, where Phe Tyr and Trp fit best
Binding these targets puts the peptide bond next to then catalytic unit

192
Q

How does trypsin bind to polypeptides to find Arg and Lys

A

Similar to chymotrypsin, picture an enzyme with a tubular hole that the AA strand can pass through, except trypsin has a narrow binding pocket and is negatively charged (attracts the +ve charge of Arg and Lys, His doesn’t fit in the narrow space)

193
Q

What is elastase

A

an enzyme with similar structure to trypsin and chymotrypsin, however has a small non-polar pocket and attracts Ala and Gly best (very small)

194
Q

What is the target of an enzyme called

A

substrate

195
Q

In order for an enzyme to react with a substrate, what must be able to occur

A

reaction must be spontaneous (enzyme only speeds it up)

196
Q

Why are non-catalyzed reactions slow

A

depends entirely on random events (molecules must collide, in right orientation, at threshold energy - these factors determine if reaction CAN occur, but doesn’t mean it will)

197
Q

What does Z represent in relation to the Arrhenius equation

A

collision frequency

198
Q

What does p represent in the Arrhenius equation

A

probability factor

199
Q

Ea represents what in the Arrhenius equation

A

activation energy

200
Q

e^(-Ea/RT) is the fraction of what in the Arrhenius equation

A

fraction of molecules at temp T which possess energy Ea

201
Q

High or low Ea and high or low temp make reaction favoured (think in terms of e^(Ea/RT))

A

lower Ea or high T make the fraction bigger, so reaction is favoured

202
Q

What is the proximity effect and which Arrenhius variable does it effect

A

enzymes bind to substrates active site and hold them close together long enough to complete reaction - this is the proximity effect

  • this increases Z in the Arrenhius equation
203
Q

What is the orientation effect and what Arrhenius variable does it effect

A

enzyme binds the substrate and holds in the active site so the reactive groups are aligned - this is the orientation effect

  • this increases p in the Arrenhius equations
204
Q

Since proximity and orientation effects are physical effects, what chemical effect can also be used to favour reaction

A

lowering Ea

205
Q

What exists at neutral pH, and at relatively low consistent temperature that would be significant to enzyme activity

A

cells - enzyme activity in cells must be done at neutral pH and fixed temp

206
Q

What is nucleophilic catalysis

A

enzymes speed up reaction by providing a better nucleophile

207
Q

What is electrophilic catalysis

A

enzyme contains a non-amino acid helper molecule called a prosthetic group that binds the enzyme to its catalytic site and initiates the reaction by withdrawing electrons from substrate

208
Q

What is general acid catalysis

A

amino acid side chain that donates H+

209
Q

What is general base catalysis

A

removes H+ from the reaction (takes H+)

210
Q

How is pH not effected by acid/base catalysis

A

catalysis occurs directly at the site of reaction, and the site of exchange is so small that it has no effect on surrounding pH

211
Q

What are the 4 types of chemical catalysis

A

nucleophilic, electrophilic, general acid, and general base

212
Q

During transition state, what can occur

A

bond stretching or change in shape which can better/worse fit a substrate

213
Q

If the active site of an enzyme is complementary to the transition state, what happens

A

less activation energy is needed

214
Q

In what 3 key ways do enzymes increase reaction rates

A
  1. physical: hold reactants close together (increase Z) and correct orientation (increase p)
  2. chemical: catalysis (nucleophilic, electrophilic, general acid, and general base)
  3. stabilizing transition state
215
Q

What values are increased in the binding step of chymotrypsin

A

Z and p (binding is physical, these are the physical ways in which reaction rate is increased)

216
Q

How does peptide hydrolysis work with H2O

A
  • H2O comes in towards peptide bond and lone pair of oxygen binds to carbon of the peptide bond
  • since carbon has 4 bonds, one of the bonds on the double bonded O becomes a lone pair on the O molecule
  • the O wants to remain double bonded, but since carbon already has 4 bonds, the nitrogen is cleaved as the leaving group with a lone pair attached
  • the peptide bond is broken

(refer to slide 3 in lectures 9-12 to better understand)

217
Q

In peptide bond hydrolysis, what acts as the nucleophile

A

oxygen on the H2O molecule (wants to give lone pair)

218
Q

In peptide bond hydrolysis, what acts as the leaving group

A

the N atom of the broken bond

219
Q

How is transition state broken down

A

reverse of peptide hydrolysis, where the bond is reformed and cleaves H2O

220
Q

How is chymotrypsin better than peptide bond hydrolysis

A

chymotrypsin does it in 2 easier steps, whereas peptide bond hydrolysis does it in 1 complicated step

221
Q

What happens in step 1 of chymotrypsin

A

nucleophilic group -X: (has lone pair) attacks the peptide C=O double bond to split off the C-terminal half of the substrate

222
Q

What is the enzyme called at the end of step one of chymotrypsin (think in terms of what has been cleaved off and what remains)

A

acyl-enzyme (intermediate)

223
Q

What does the acyl-enzyme act as for step 2 of chymotrypsin

A

the substrate

224
Q

What happens in step 2 of chymotrypsin

A

brings in H2O to release the N-terminal half and restore enzyme group -X: to its original state

225
Q

How many chymotrypsin reactions can occur per second

A

40 (fast)

226
Q

How many reactions occur for peptide hydrolysis using water

A

1 reaction in 10 years (slow)

227
Q

Chymotrypsin uses a better nucleophile compared to peptide hydrolysis, in the form of the _________ __________

A

catalytic triad

228
Q

What is the catalytic triad in chymotrypsin

A

three amino acids that line up side by side in correctly folded chymotrypsin and cooperate for maximum effect

Asp 102, His 57, and Ser 195

229
Q

Why is His able to act as both an acid and a base

A

because it has a pKa of 6.5 which means at standard conditions it is not in its fully protonated or deprotonated form - it can act as both

230
Q

What is enzyme assay

A

the process of measuring enzyme-catalyzed reaction rate

231
Q

What is enzyme kinetics

A

mathematical analysis of how rate varies as a function of substrate concentration (how does concentration affect rate of reaction)

232
Q

What is a better way to measure enzyme in reaction

A

artificial substrates
- they are molecular look alikes for the actual substrate

233
Q

Some natural substrates show colour, while others show no colour but show ____ ____________ _________

A

UV absorbance change

234
Q

Coloured or UV-absorbing molecules contain ______________

A

chromophores

235
Q

What are chromophores

A

parts of molecules with conjugated double bonds

236
Q

What law measures absorbance

A

Beer Lambert Law

237
Q

What is Beer Lambert Law

A

A=elc

238
Q

What are the variables in Beer Lambert law

A

A = absorbance
e = extinction coefficient
l = length
c = concentration

239
Q

Does absorbance have units

A

NO

240
Q

What is the equation for rate of reaction

A

[ ] / time

241
Q

What is the equation for enzyme activity

A

rate x volume

242
Q

What does enzyme activity represent

A

the quantity of enzyme present

243
Q

What is the equation for specific activity

A

enzyme activity / total protein

244
Q

What does the specific activity represent

A

the purity of enzyme

245
Q

What is the equation for molar activity

A

specific activity x molar mass

246
Q

What is the equation for total enzyme

A

[E] + [ES] = total enzyme

247
Q

What is the equation (theoretically) for [E]

A

how much of the enzyme is empty or unoccupied

248
Q

What is the equation (theoretically) for [ES]

A

how much of the enzyme is full/occupied

249
Q

Define turnover number

A

the amount of substrate in moles being catalyzed per mol of enzyme per second

250
Q

What is the equation for turnover number

A

specific activity x molar mass

251
Q

Do enzyme reactions follow simple rate laws

A

no

252
Q

Since enzymes are not consumed, what do we know about [E]total

A

it is constant

253
Q

At steady state, how are rate of breakdown and rate of formation related

A

they are equal

254
Q

What is the Michaelis-Menten equation

A

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

255
Q

When 100% of enzyme present is occupied by substrate, what do we get

A

Vmax = K2 [E]total

256
Q

What is the Michaelis constant

A

Km

257
Q

What is the Michaelis-Menten equation in fraction form

A

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

258
Q

What variable is the upper limit for rate

A

Vmax

259
Q

What does Vmax represent

A

the catalytic rate when 100% of the enzyme is occupied by substrate

260
Q

What does a higher Vmax mean

A

higher Vmax = faster catalytic rate

261
Q

What does pseudo-constant mean

A

dependant on the amount of enzyme present (so it is constant only if the amount of enzyme is fixed)

262
Q

What is the true constant in Vmax = K2[E]total

A

K2 (the turnover number)

263
Q

What does Km represent

A

the concentration of substrate at half Vmax
- indicates how well a substrate binds to the catalytic site

264
Q

What does a low Km indicate

A

enzyme binds and utilizes substrate well

265
Q

What does a high Km indicate

A

enzyme binds and utilizes substrate poorly

266
Q

What is the lineweaver burk method

A

linear transformations convert the Michaelis Menten equation into straight line form

267
Q

How does the lineweaver burk method alter the equation

A

take reciprocals of both sides
1/Vo = Km + [S] / Vmax

268
Q

According to the lineweaver burk method, what is the equation for slope

A

Km/Vmax

269
Q

According to the lineweaver burk method, what is the equation for y-int

A

1/Vmax

270
Q

According to the lineweaver burk method, what is the equation for x-int

A

-1/Km

271
Q

According to the lineweaver burk method, what is the equation for the y axis

A

1/Vo

272
Q

According to the lineweaver burk method, what is the equation for the x axis

A

1/[S]

273
Q

How do inactivators interact with enzymes

A

irreversibly

274
Q

How do inhibitors interact with enzymes

A

reversibly

275
Q

How do inactivators chemically bind to enzymes

A

covalently

276
Q

How do inhibitors chemically bind to enzymes

A

non-covalently: instead binds to site on enzyme similar to how a substrate would

277
Q

What does competitive inhibition affect

A

ability to bind to substrate

278
Q

What does no-competitive inhibition affect

A

catalytic rate

279
Q

What is a real world general example of enzyme inhibitors

A

many drugs are enzyme inhibitors

280
Q

How does competitive inhibition work

A

inhibitor and the substrate compete for available enzyme

281
Q

How is competitive inhibition overcome

A

when [S] is high, competitive inhibition can be overcome (because there is enough substrate to overpower the inhibitor)

282
Q

Ki is the concentration of inhibitor [I] that causes…

A

Km to double

283
Q

On a linewearver burk plot, what point do all [S] lines have in common

A

share the same y-int
(this is because Vmax is not changed by competitive inhibition)

284
Q

On a lineweaver burk plot of competitive inhibition, as the Km value increases so does [I], so on the plot…

A

as Km and [I] increase, x-int gets smaller (closer to 0)

285
Q

What is non-competitive inhibition

A

when the substrate and inhibitor can both bind to the enzyme on different sites, but not yeild a product

286
Q

If EI and EIS steps have a different Ki what occurs

A

mixed inhibition

287
Q

What happens to Vmax as [I] increases in non-competitive inhibition

A

Vmax decreases

288
Q

What stays constant in non-competitive inhibition

A

Km

289
Q

Ki is the concentration of inhibitor [I] that causes Vmax to…

A

halve

290
Q

On a lineweaver burk plot of non-competitive inhibition, what do all [I] lines have in common

A

share same x-int

291
Q

On a lineweaver burk plot of non-competitive inhibition, as the Vmax value decreases [I] increases, so on the plot…

A

the y-int gets larger (y-int = 1/Vmax)

292
Q
A

Bioc 2580 (16 decks)

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