Midterm Study Set Flashcards
What is the protein that stores oxygen in muscle tissue
myoglobin
What measurement is protein size measured in
kilodaltons
What are the building blocks of proteins
amino acids
What bond holds amino acids together
peptide bonds
What is the positive end of an amino acid structure
amino group
What is the negative end of an amino acid structure
carboxylate group
What differentiates the 20 amino acids
the R groups
What are the 2 possible spatial arrangements of amino acids
L and D
For stereoisomers, what is the term for mirror images (D and L configurations)
enantiomers
What configuration are proteins found in
L configuration: amine group of the left (L for left)
What is a condensation reaction
cleavage of H2O
What is a hydrolysis reaction
using H2O to form a bond
Where is the point of weakness on a peptide bond where H2O can form/break bonds
the C=O bond
In which direction does the equilibrium of the reaction lie under standard biochemical conditions (in terms of hydrolysis and condensation)
hydrolysis
In order for the reaction of a peptide to move in the direction of peptide bond synthesis, what must occur
carboxyl group needs to be chemically modified
How are polypeptides and oligopeptides different
polypeptides = many amino acids
oligopeptides = a few amino acids
Why are the amino acids in a chain known as amino acid residues
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
How can the number of amino acid residues be estimated given the molecular weight of a protein
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
What carbon is the central carbon in the backbone (think in terms of greek letters)
alpha carbon
What is the order of carbons in an amnio acid chain (think in terms of greek letters)
alpha, beta, gamma, delta, epsilon
What are the 3 properties to consider between the different amino acid side chains
polarity, charge, and H-bond ability
What are the 6 very non polar amino acids
Alanine, Valine, Leucine, Isoleucine, Methionine, and Phenylalanine
What are the 5 non polar amino acids
Glycine, Cysteine, Proline, Tyrosine, and Tryptophan
What are the 4 uncharged but polar amino acids
Serine, Threonine, Asparagine, and Glutamine
What are the 3 positively charged amino acids
Lysine, Histidine, and Arginine
What are the 2 negatively charged amino acids
Aspartate and Glutamate
If an amino acid is non-polar is it hydrophilic or hydrophobic
hydrophobic
If an amino acid is polar is it hydrophilic or hydrophobic
hydrophilic
Why do polar molecules interact with water
polar attracts polar, and water is a polar molecule as well
What is the sequence of electronegativity of elements found in amino acid chains
O>N>S>C=H (O being most electronegative, C and H being least)
Why would an atom with a longer CH chain be considered more non-polar
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
What about Serine and Threonine make them polar but still uncharged
the hydroxyl group on the R chain
What about Aspirigine and Glutamine make them polar but still uncharged
the amide group on the R chain
What are good examples of hydrogen donors for H-bonds
OH- or NH- groups
What are good examples of hydrogen acceptors
O or N atoms with lone pair of electrons
What about Lysine, Histidine, and Arginine make them positively charged and polar
the NH3+/NH2+/NH+ groups on the R chain
What about Aspartate and Glutamate make them negatively charged
deprotonated R chains with a carboxylate group (COO-)
What are the 7 AAs that can donate/accept a proton on its R chain
Aspartate, Glutamate, Tyrosine, Cysteine, Arginine, Histidine, Lysine
What are the 4 AAs that go from neutral to negatively charged when deprotonated
Aspartate, Glutamate, Tyrosine, and Cysteine
What are the 3 AAs that go from positive to neutral when deprotonated
Lysine, Arginine, and Histidine
Henderson-Hasselbalch equation
pH = pKa + log(deprotonated/protonated)
Why are amino acids considered dipolar
has both a +ve and -ve charge (NH3+ and COO-)
What happens when pKa=pH
50% of AA is in protonated form and 50% is deprotonated
Why does deprotonation occur with raised pH
[H+] becomes less available so deprotonation is more likely to occur
If the pH is one or more units below the pKa, the AA is…
protonated
If the pH is one or more units above the pKa, the AA is…
deprotonated
If the pH is less than one unit above or below the pKa then what happens…
calculation is needed
What is amino acid analysis and what two processes does it involve
functions that help determine protein structure
- involves separation and detection
What is partition chromatography (column chromatography)
stationary phase resides in a column and a mobile phase is run through the column to separate amino acid proteins
How are amino acids measured from column chromatography
concentration of protein(s) is measured in the elution volume
What is thin layer chromatography
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
Which proteins travel further in gel filtration
non-polar amino acids
What is the stationary and mobile phases of gel filtration
stationary = plastic sheet
mobile = solvent buffer
What is added to gel filtration that indicates proteins present
ninhydrin
Ion chromatography separates based on __________
charge
How does a cation exchange resin work
contain negative groups within the resin, so bind to positive groups in sample
How does an anion exchange resin work
contain positive groups within the resin, so bind to negative groups in sample
What determines how tight proteins bind to an ion exchange resin
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)
Where are amino acid concentrations measured in ion exchange chromatography
the elution volume
What is affinity chromatography
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
What is a “tag” in relation to a ligand
a peptide/protein that is fused to the target protein and is capable of binding to a ligand
What is metal affinity chromatography
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
What is a benefit of metal affinity chromatography
high purification in minimal steps
What characteristic does gel filtration chromatography seperate proteins based on
allows separation of proteins based on size
How does gel filtration chromatography work
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
What is gel electrophoresis
sample is injected into a gel and the proteins move along the gel based on charge
What is SDS PAGE
sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)
How does SDS PAGE work
proteins are treated with SDS, so all have same uniform charge, separation is based solely on size
- smaller migrate faster than large
What is isoelectric focusing
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
What are two dimensional gels
combine SDS PAGE and isoelectric focusing
What is mass spectrometry
protein is vaporized by a laser beam and particles travel toward the detector
- velocity at which the particles travel depends on mass
How to determine the protein in sample in mass spectrometry
using a database of protein masses
What is an enzyme unit
the amount of enzyme that converts one umol of substrate to product per min
What is enzyme activity
total units of enzyme present in a solution
What is the specific activity of an enzyme
number of enzyme units per milligram of total protein
What is the equation for specific activity
specific activity = (enzyme activity/total protein)
With purification, does specific activity increase or decrease
specific activity increases with each purification step (based on the equation you’d be dividing by smaller values)
When samples of pure and unpure versions of the SAME enzyme are examined, what can be determined
enzyme purity
With purification of a protein, what happens to enzyme activity
decreases
With purification of a protein, what happens to total protein
decreases
With purification of a protein, what happens to total protein
increases
How are amino acids separated from a chain to begin with
hydrolysis of peptide bonds
What is a nucleophile
an atom with a lone pair of electrons available to share
What is an electrophile
a lone pair seeking atom (wants an atom to come bond with it)
How can an atom with a lone pair use it
- by h-bonding
- as a base (captures [H+])
- as a nucleophile
What is nucleophilic substitution
incoming nucleophile attacks the target atom to displace the leaving group (leaving group takes bonding electrons with it)
What is nucleophilic addition
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
Are carbon-carbon bonds good or bad leaving groups
bad; C-C bonds are hard to break so they are poor leaving groups
How many helical segments are in myoglobin
8
What is primary structure in a protein
the linear sequence of amino acids
What is secondary structure of a protein
the repetitive patterns of the peptide chain (ie. helices or pleated sheets)
What is the tertiary structure of a protein
the overall 3D pattern of a single polypeptide
What is quaternary structure of a protein
combining multiple polypeptides to form a protein complex
What scientist worked with insulin and developed the method of investigating proteins using fluorodinitrobenzene
Fred Sanger
How does fluorodinitrobenzene work to investigate proteins
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
What is the downfall to fluorodinitrobenzene as an investigative method of protein
can only investigate the first amino acid of the sequence (hydrolysis destroys the rest of the chain)
Who improved the Sanger method
Perh Edman (Edman degradation)
What is the main difference between the Sanger method and Edman degradation
Edman degradation can be repeated and is done without hydrolyzing the rest of the chain
What are the two steps of Edman degradation
coupling and cyclization
What does coupling require in Edman degradation
base
What does cycling require in Edman degradation
acid
How many times can the Edman degradation cycle be repeated
up to 50 times
What is the purpose of selective hydrolysis
to cut long protein chains into smaller oligopeptides (divide and conquer type method)
What does the digestive enzyme trypsin bind and recognize
Arg and Lys
What is the exception to the binding and cutting of Arg and Lys
cannot occur when proline is the following AA
What is the positioning of Arg/Lys to trypsin when the protein is cut
the carboxylate end of Arg or Lys is positioned at the catalytic site of trypsin
WHY does hydrolysis of Arg or Lys not work when followed by proline
proline doesn’t fit correctly into the catalytic site which prevents the ability to cut
What does chymotrypsin act on
tyrosine, tryptophan, and phenylalanine
WHY does the hydrolysis of Tyr, Trp, and Phe not work when followed by proline
similarly to the previously stated with trypsin, the proline AA does not fit into the catalytic site of chymotrypsin either
What does cyanogen bromide act on
chemical reagent that acts on methionine residues
How does cyanogen bromide work
acts on methionine residues and turns methionine into homoserine (Hse)
What is the overlap method
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
How are most amino acid sequences derived now
using DNA sequences to then predict the original amino acid sequence
What is tandem mass spectrometry
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
What is the protease of choice in mass spectrometry
trypsin
How does trypsin play importance to mass spectrometry
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
How to find mass of amino acid using data from mass spectrometry
difference in mass from largest peak to next largest peak gives mass of unknown AA (calculate backwards)
What is BLAST searching
basic local alignment search tool
- compares input sequence to a database to confirm protein sequences or discover new ones
How is the backbone of a polypeptide chain flexible
bond rotation
How does bond rotation work
groups connected by single bonds can rotate about a bond axis
Do bonds bend?
NO
What are conformations
represent states of a molecule that can be interconverted by bond rotations without breaking covalent bonds
What are configurations
can only be interchanged by breaking covalent bonds, not by rotation
What are examples of configurations of molecules
cis and trans
What are the two chiral configurations of amino acids
L and D
What does X-ray diffraction measure
regular repeating patterns on the molecular scale
- dimensions of the repeating pattern can be calculated
Who interpreted the fibre patterns in fibrous proteins
Linus Pauling
What was Paulings key finding
peptide bond has double bond character
How did Pauling determine his conclusions
length of a peptide bond was closer to that of a double bond than a single bond, therefore it has more double bond character
Is a peptide bond flexible or rigid
rigid and fixed
Why is a peptide bond rigid
because it has double bond character
Within restricted bond rotation, what are the possibilities for structure and how are they arranged
helical: a-c bond down peptide chain turns in same direction
extended: a-c bond turns in alternate direction
no shape: random coil
How many amino acids occur per turn of the alpha helix
3.6 amino acids
How many amino acids are required minimum to create an alpha helix
5
What is the distance between each turn of the alpha helix
5.4 Armstrongs
What is the distance along the alpha helix per amino acid
1.5 Armstrongs
Where is the hydrogen bond found in the alpha helix that holds its shape together
- C=O lines up with 5. H-N
(2. C=O lines up with 6. H-N, etc.)
In relation to beta sheets, strands in opposing directions form _______________ B-sheet, whereas strands in same direction form _________________ B-sheet
antiparallel
parellel
Which amino acids prefer beta pleated sheet orientation
Trp, Tyr, Phe (all big in size), Val, Ile, Thr (have a branch on B-C), and Cys (large S atom on B-C)
What determines the overall shape of an AA strand
local majority - does the majority prefer alpha helix or beta sheet orientation
What are the 5 breakers of secondary structure
GPNDS (glycine, proline, arginine, aspirigine, and serine
How many breakers are required in what amount of space to effectively work
2 breakers in a group of 4 amino acids
What is the point of a breaker
forms a flexible loop or turn and allows the polypeptide chain to change direction drastically
What is denaturation
when a protein unfolds as a result of unnatural environmental factors
What are ways to denature a protein
heat, disruptive solvents, SDS
What is the simplest form of tertiary structure of polypeptide
no breakers; no rotation
What has a unique triple helix structure in our bodies
collagen
Since the structure of proteins is rigid, they are fibrous, what does this mean?
not able to fold
If a-keratin is fibrous, what does this mean about its structure
it has no breakers
What amino acids are enclosed towards the middle
non-polar
What amino acids are on the exterior of the folding
polar
Why are non-polar amino acids on the interior of folding pattern
because of the hydrophobic effect, want to minimize contact with H2O
Why are polar amino acids found on the exterior of folding pattern
interact well with H2O (good H-bonding)
What forces are at play when proteins fold together in a jigsaw puzzle effect (tightly interlocked)
weak van der Waal forces by close contact
With a sequence of mostly a-helices, what overall structure will form
a-helix bundle
What creates a non-polar patch/stripe
non-polar amino acids every 3 or 4 places in an AA sequence that fold inside the bundle
What is more stable; antiparallel or parallel beta sheets
antiparallel: H-bonds are arranged in a straight line (diagonal in parallel)
When a sheet is polar on one side and non-polar on the other side, what happens
the sheet folds with non-polar facing inwards to form a beta barrel
Sequences with alternating alpha and beta configuration can form what
a parallel beta sheet
Where do the helices lie on a parallel sheet
above or below the plane of the sheet
Why are parallel pleated sheets less stable
the H-bonds run on a diagonal
If parallel pleated sheets are usually buried in the centre of a protein, what kind of amino acids is it typically made of
non-polar
If all helices lie on the same side of a parallel sheet, what configuration does it take on
alpha beta barrel
What is an alpha beta barrel
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
If the helices on a parallel sheet lie on both sides of the sheet, what configuration does it take on
alpha beta sandwich
What is an alpha beta sandwich
a parallel beta-sheet where alpha-helices lie on both sides of the sheet (think sheet is sandwiched by helices)
What are domains in respect to large proteins
larger proteins fold up into sections called domains (ie. a protein might have 2 ab sandwich domains)
What is the native state of a protein
its normal, folded state
What is the term for when a protein folds and loses function
denaturation
Where is covalent bonding found in proteins
covalent bonding links amino acids (peptide bonds are covalent)
Where us non-covalent bonding found in proteins
dictate the folding pattern and stability
(most importantly the hydrophobic effect and van der Waals forces)
What are the most important non-covalent bonding features of proteins
hydrophobic effect and van der Waals forces
How much total energy of native state does the hydrophobic effect contribute
50%
What is another term for van der waals forces
London dispersion forces
What are van der Waals forces
weak electrostatic forces between atoms that are close in proximity
What is the important note regarding van der Waals forces in proteins
need many atoms in close contact to actually have an effect, since they are weak interactions
How is a salt bridge formed
negative side chains pair up to positive side chains nearby and create a salt bridge in the folded protein
Do ion pairs and H-bonding contribute more or less to tertiary protein folding compared to hydrophobic or van der Waals interactions
less impact, van der Waals and hydrophobic interactions have a greater effect
Why do van der waal forces and the hydrophobic effect have a greater impact on protein folding
because polar AAs face the exterior and these forces target polar AAs
What forces have greatest effect on secondary protein folding
H-bonds
What kind of bonds help hold tertiary structure of proteins
disulphide bonds
How do disulphide bonds form in tertiary protein structure
pairs of SH groups (from cysteine AAs) react with O2 and release H2O
Are disulphide bonds common in proteins
no; only few have them and those are likely proteins that function outside cells (since O2 is needed)
What is the experiment done by Christian Anfinsen
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
Non polar patches on proteins bind by what effect
hydrophobic effect
Matching shapes on proteins bind by what effect
van der Waals forces (maximized close contact)
Match of charged groups bind by what effect
H-bond effect (or just charges)
What do enzymes recognize and bind
enzymes recognize and bind specific target proteins and catalyze reactions
What do antibodies recognize and bind
bind and identify foreign molecules in the body
What amino acids fit best in the chymotrypsin binding pocket
Phe, Tyr, and Trp
How does chymotrypsin bind to polypeptides to find Phe, Tyr, and Trp
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
How does trypsin bind to polypeptides to find Arg and Lys
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)
What is elastase
an enzyme with similar structure to trypsin and chymotrypsin, however has a small non-polar pocket and attracts Ala and Gly best (very small)
What is the target of an enzyme called
substrate
In order for an enzyme to react with a substrate, what must be able to occur
reaction must be spontaneous (enzyme only speeds it up)
Why are non-catalyzed reactions slow
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)
What does Z represent in relation to the Arrhenius equation
collision frequency
What does p represent in the Arrhenius equation
probability factor
Ea represents what in the Arrhenius equation
activation energy
e^(-Ea/RT) is the fraction of what in the Arrhenius equation
fraction of molecules at temp T which possess energy Ea
High or low Ea and high or low temp make reaction favoured (think in terms of e^(Ea/RT))
lower Ea or high T make the fraction bigger, so reaction is favoured
What is the proximity effect and which Arrenhius variable does it effect
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
What is the orientation effect and what Arrhenius variable does it effect
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
Since proximity and orientation effects are physical effects, what chemical effect can also be used to favour reaction
lowering Ea
What exists at neutral pH, and at relatively low consistent temperature that would be significant to enzyme activity
cells - enzyme activity in cells must be done at neutral pH and fixed temp
What is nucleophilic catalysis
enzymes speed up reaction by providing a better nucleophile
What is electrophilic catalysis
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
What is general acid catalysis
amino acid side chain that donates H+
What is general base catalysis
removes H+ from the reaction (takes H+)
How is pH not effected by acid/base catalysis
catalysis occurs directly at the site of reaction, and the site of exchange is so small that it has no effect on surrounding pH
What are the 4 types of chemical catalysis
nucleophilic, electrophilic, general acid, and general base
During transition state, what can occur
bond stretching or change in shape which can better/worse fit a substrate
If the active site of an enzyme is complementary to the transition state, what happens
less activation energy is needed
In what 3 key ways do enzymes increase reaction rates
- physical: hold reactants close together (increase Z) and correct orientation (increase p)
- chemical: catalysis (nucleophilic, electrophilic, general acid, and general base)
- stabilizing transition state
What values are increased in the binding step of chymotrypsin
Z and p (binding is physical, these are the physical ways in which reaction rate is increased)
How does peptide hydrolysis work with H2O
- 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)
In peptide bond hydrolysis, what acts as the nucleophile
oxygen on the H2O molecule (wants to give lone pair)
In peptide bond hydrolysis, what acts as the leaving group
the N atom of the broken bond
How is transition state broken down
reverse of peptide hydrolysis, where the bond is reformed and cleaves H2O
How is chymotrypsin better than peptide bond hydrolysis
chymotrypsin does it in 2 easier steps, whereas peptide bond hydrolysis does it in 1 complicated step
What happens in step 1 of chymotrypsin
nucleophilic group -X: (has lone pair) attacks the peptide C=O double bond to split off the C-terminal half of the substrate
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)
acyl-enzyme (intermediate)
What does the acyl-enzyme act as for step 2 of chymotrypsin
the substrate
What happens in step 2 of chymotrypsin
brings in H2O to release the N-terminal half and restore enzyme group -X: to its original state
How many chymotrypsin reactions can occur per second
40 (fast)
How many reactions occur for peptide hydrolysis using water
1 reaction in 10 years (slow)
Chymotrypsin uses a better nucleophile compared to peptide hydrolysis, in the form of the _________ __________
catalytic triad
What is the catalytic triad in chymotrypsin
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
Why is His able to act as both an acid and a base
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
What is enzyme assay
the process of measuring enzyme-catalyzed reaction rate
What is enzyme kinetics
mathematical analysis of how rate varies as a function of substrate concentration (how does concentration affect rate of reaction)
What is a better way to measure enzyme in reaction
artificial substrates
- they are molecular look alikes for the actual substrate
Some natural substrates show colour, while others show no colour but show ____ ____________ _________
UV absorbance change
Coloured or UV-absorbing molecules contain ______________
chromophores
What are chromophores
parts of molecules with conjugated double bonds
What law measures absorbance
Beer Lambert Law
What is Beer Lambert Law
A=elc
What are the variables in Beer Lambert law
A = absorbance
e = extinction coefficient
l = length
c = concentration
Does absorbance have units
NO
What is the equation for rate of reaction
[ ] / time
What is the equation for enzyme activity
rate x volume
What does enzyme activity represent
the quantity of enzyme present
What is the equation for specific activity
enzyme activity / total protein
What does the specific activity represent
the purity of enzyme
What is the equation for molar activity
specific activity x molar mass
What is the equation for total enzyme
[E] + [ES] = total enzyme
What is the equation (theoretically) for [E]
how much of the enzyme is empty or unoccupied
What is the equation (theoretically) for [ES]
how much of the enzyme is full/occupied
Define turnover number
the amount of substrate in moles being catalyzed per mol of enzyme per second
What is the equation for turnover number
specific activity x molar mass
Do enzyme reactions follow simple rate laws
no
Since enzymes are not consumed, what do we know about [E]total
it is constant
At steady state, how are rate of breakdown and rate of formation related
they are equal
What is the Michaelis-Menten equation
Vo = Vmax [S] / Km + [S]
When 100% of enzyme present is occupied by substrate, what do we get
Vmax = K2 [E]total
What is the Michaelis constant
Km
What is the Michaelis-Menten equation in fraction form
Vo/Vmax = [S] / Km + [S]
What variable is the upper limit for rate
Vmax
What does Vmax represent
the catalytic rate when 100% of the enzyme is occupied by substrate
What does a higher Vmax mean
higher Vmax = faster catalytic rate
What does pseudo-constant mean
dependant on the amount of enzyme present (so it is constant only if the amount of enzyme is fixed)
What is the true constant in Vmax = K2[E]total
K2 (the turnover number)
What does Km represent
the concentration of substrate at half Vmax
- indicates how well a substrate binds to the catalytic site
What does a low Km indicate
enzyme binds and utilizes substrate well
What does a high Km indicate
enzyme binds and utilizes substrate poorly
What is the lineweaver burk method
linear transformations convert the Michaelis Menten equation into straight line form
How does the lineweaver burk method alter the equation
take reciprocals of both sides
1/Vo = Km + [S] / Vmax
According to the lineweaver burk method, what is the equation for slope
Km/Vmax
According to the lineweaver burk method, what is the equation for y-int
1/Vmax
According to the lineweaver burk method, what is the equation for x-int
-1/Km
According to the lineweaver burk method, what is the equation for the y axis
1/Vo
According to the lineweaver burk method, what is the equation for the x axis
1/[S]
How do inactivators interact with enzymes
irreversibly
How do inhibitors interact with enzymes
reversibly
How do inactivators chemically bind to enzymes
covalently
How do inhibitors chemically bind to enzymes
non-covalently: instead binds to site on enzyme similar to how a substrate would
What does competitive inhibition affect
ability to bind to substrate
What does no-competitive inhibition affect
catalytic rate
What is a real world general example of enzyme inhibitors
many drugs are enzyme inhibitors
How does competitive inhibition work
inhibitor and the substrate compete for available enzyme
How is competitive inhibition overcome
when [S] is high, competitive inhibition can be overcome (because there is enough substrate to overpower the inhibitor)
Ki is the concentration of inhibitor [I] that causes…
Km to double
On a linewearver burk plot, what point do all [S] lines have in common
share the same y-int
(this is because Vmax is not changed by competitive inhibition)
On a lineweaver burk plot of competitive inhibition, as the Km value increases so does [I], so on the plot…
as Km and [I] increase, x-int gets smaller (closer to 0)
What is non-competitive inhibition
when the substrate and inhibitor can both bind to the enzyme on different sites, but not yeild a product
If EI and EIS steps have a different Ki what occurs
mixed inhibition
What happens to Vmax as [I] increases in non-competitive inhibition
Vmax decreases
What stays constant in non-competitive inhibition
Km
Ki is the concentration of inhibitor [I] that causes Vmax to…
halve
On a lineweaver burk plot of non-competitive inhibition, what do all [I] lines have in common
share same x-int
On a lineweaver burk plot of non-competitive inhibition, as the Vmax value decreases [I] increases, so on the plot…
the y-int gets larger (y-int = 1/Vmax)
