MT 1 Flashcards
Biochemistry
The chemical substances and vital processes occurring in a living organism
Cellular metabolism (aka chemical rxns in a cell)
Lite’s wiring diagram → dots biomolecules
Biomolecule: organic compound normally present as an essential compound of living organisms
This figure looks complex because all of the pathways are connected
List the types of biomolecules
- Carbohydrates (sugars)
- Lipids
- Proteins
- Nucleic acids
Carbohydrates (sugars) Functions
Energy and energy storage (glucose & glycogen)
Cell recognition (glycosylation)
Structural (ie. in plants, cellulose)
Component of DNA (deoxyribose) and RNA (ribose)
Lipids functions
Energy and energy storage (triglycerides [TG], fats, fatty acids)
Structures/barrier (ie. membranes)
Signalling (steroid hormones)
Insulation (blubber)
Proteins functions
Catalysis (enzymes: lactase, alcohol dehydrogenase)
Signalling (hedgehog, ubiquitin, insulin)
Structure (collagen, histone)
Transport (membrane transporters, hemoglobin, LDL)
Defense (antibodies)
Storage (ferritin)
Movement (actin/myosin)
Synthesis (protein, DNA synthesis)
Nucleic acids functions
Information (DNA/RNA)
Energy (ATP, GTP)
Transport (tRNAs) ← beyond scope of the course
Catalysis (ribosomes)
Components of cofactors (NAD, FAD)
Most biomolecules are composed of…
Carbon
Hydrogen
Oxygen
Nitrogen
Phosphorus (nucleic acid & ATP/GTP)
Sulfur
Others too!
We study how biomolecules […].
These […] in biomolecules are known as […].
We study how biomolecules interact with each other and themselves. These interactions between elements in biomolecules are known as bonding.
List the types of bonding
- Covalent bonds
- Ionic bonds
- Hydrogen bonds
- Van der Waal interactions
- Hydrophobic interactions
Covalent bonds
Sharing of electrons between 2 adjacent atoms
Drawn as solid lines
High energy
Not easily reversible (stable)
Relatively shorter (smaller bond length)
Bind together elements in biomolecules
Geometry of carbon bonding
When carbon has 4 single bonds, it adapts tetrahedral structure, with bonds between carbons at 109 degrees with free rotation around each bond.
When carbon has a double bond, with trigonal (flat) planar structure with 120 degree angle → single bonds in same plane → 1 double bond, 2 single bonds
Triple bonds not important for biomolecules
Ionic bonds
Interaction of two charged atoms/particles
Described by Coulomb’s law: F = q1q2/E*r2
What is E in Coulomb’s law?
E is dielectric constant; takes into account medium where interaction takes place. H2O has the highest dielectric constant, thus lowering the force of interaction. Electrostatic interactions determine helical structure of DNA
Hydrogen bonds
Definition: Hydrogen atom that is partially charged by electronegative atom
H-bond requires H-donor (with H-covalently bound to it) and H-acceptor (which has a lone pair of e-).
Both hydrogen acceptors and donors are usually oxygen and nitrogen (sometimes sulfur)
It is based on electrostatic interaction; electronegative donor tends to pull e- away from hydrogen. As a result, donor becomes partly negative and hydrogen becomes partly positive
Hydrogen bonds are weak (4-15 kjol/mole) and longer (relative to covalent or ionic)
Van der Waals Interactions
Attraction of two molecules
At any given time, charge distribution around an atom is not symmetric
This asymmetry causes complimentary asymmetry on other atoms, leading to attraction
Has small energy
If the atoms get too close, they repel
There is a “sweet spot”
Water in biochemistry
Almost all reactions in the body happen in aqueous solution
H2O has a huge effect on reactions
H2O molecule is bent and can form multiple H-bonds
H2O molecules form H-bonds with each other
Based on water solubility, biomolecules can be divided into 3 groups. List them.
Hydrophilic, hydrophobic, amphipathic
Hydrophilic
Water soluble
Polar or charged (ie. NaCl)
Hydrophobic
Not soluble in water (ie. fats, oils)
Amphipathic
Molecules that containboth hydrophilic and hydrophobic parts (ie. tryptophan, tyrosine, lysine, methionine)
Very often, water needs to be […] to allow various […] to occur because […].
Very often, water needs to be excluded or manipulated to allow various electrostatic interactions to occur (ie. catalyst)
Water will disrupt hydrogen bonding
The Laws of Thermodynamics
- Total energy of a system and its surroundings is constant. In other words, you don’t create or destroy energy; you can only change its form
- Total entropy (S=entropy=measure of randomness) of a system and its surroundings always increases for a spontaneous process. But entropy can decrease locally (ie. complimentary strands of DNA) but heat will be released, so 2nd law is still true.
Gibb’s Free Energy Equation and implications of ∆G, ∆H, ∆S
∆Gsys = ∆H - T∆Ssys
Where:
∆G = Gibb’s free energy (kJ/mole)
T = Temperature in K
If ∆G < 0, the reaction is spontaneous (exergonic)
If ∆G > 0, the reaction is non-spontaneous (endergonic)
∆H < 0, the reaction releases heat => ∆G is more negative => more spontaneous
∆S > 0 => more disorganized => ∆G is more negative => more spontaneous
What drives hydrophobic interactions?
When a non-polar molecule is added to H2O, the water molecules are forced into a shell. This lowers entropy. However, with time, non-polar molecules come together and H2O molecules form a shell only at the edge, and entropy increases.
pH, buffers, Kw
Many biomolecules can act as weak acids and bases
Behaviour of biomolecules depends on ionization state, which is determined by pH
Because pH is important, it must be maintained at a certain level with buffers.
pH: A measure of concentration of H+ in solution
Buffer: A mixture of weak acid and conjugate base. It resists changes in pH. Buffering region is usually 1 pH unit on either side of pKa.
pH = -log[H+]
Scale = 0-14, where 0 is a strong acid and 14 is a strong base
H2O ⇌ H+ + OH-
Kw = ionization constant = 1 * 10-14
Kw = [H+][OH-]
Weak acid and bases review, Ka and pKa
Weak acids and bases don’t fully ionize in solution
CH3OOH ⇌ CH3OO- + H+
Ka = [A-][H+] / [HA] = [CH3OO-][H+] / [CH3OOH]
pKa = -log[Ka]
Henderson-Haselbach equation
If we titrate a weak acid with a strong base (NaOH), we can calculate pH using Henderson-Haselbach equation
pH = pKa + log[A- / HA]
If pH = pKa, [HA] = [A-]
If pH < pKa, [HA] > [A-]
If pH > pKa, [HA] < [A-]
There is a region where pH doesn’t change much => buffering region
List the Three Key Buffers in Biological Systems
- Carbonate/Bicarbonate Buffer
- Phosphate Buffer
- Histidine and cysteine
Carbonate/Bicarbonate Buffer
CO2(dissolved) + H2O ⇌ H2CO3 (carbonic acid) ⇌ H+ + HCO3- (bicarbonate ion)
Phosphate Buffer
H2PO4- (dihydrogen phosphate ion) ⇌ H+ + HPO42- (monohydrogen phosphate ion)
Proteins
Linear polymers built out of α-amino acids (αα)
Proteins final 3D shape and function depends on its sequence of αα
Each αα has different functional groups, allowing for massive diversity
Proteins can be flexible or rigid
Proteins can interact with each other
Amino Acids
Contain central carbon (α-carbon), attached to amino group, carboxylic acid group, hydrogen atom, and unique side chain (R)
Note: α-carbon is chiral
Chiral center/Enantiomers/L vs D
Chiral center: Atom with its substituents arranged so that the molecule is NOT superimpossible on its mirror image
This means that there are 2 enantiomers for each amino acid (except glycine)
Enantiomer: pair of molecules, each with one or more chiral centre that are mirror images of each other
If the amino group is on the left, it is in the L-form (otherwise D-form)
In biological systems, only L-aa’s exist in proteins and all living things
At pH = 7, all amino acids exist in _______
At pH = 7, all aa’s exist in zwitterion
Zwitterion: ion with both (+) and (-) charge
What determines amino acids variability?
The side chains (R roups)
Glycine
Gly, G
No chiral carbon
Technically, not really hydrophobic
Isoleucine
Aliphatic
Has a second chiral center
Methionine
Aliphatic
Contains thio-ether (-S-C) group
Proline
Contains a ring; changes 3D structure of amino acid
Still aliphatic
Twist in side chain; ring structure makes it more rigid/more restrained
Often introduces kinks in amino acid polypeptide chain
Nonpolar, aliphatic amino acids
Aliphatic: Open chain structure (alkanes)
- Glycine, Gly, G
- Alanine, Ala, A
- Valine, Val, V
- Leucine, Leu, L
- Isoleucine, Ile, I
- Methionine, Met, M
- Proline, Pro, P
All of these are hydrophobic, often found in the center of a protein or in memebrane crossing domain
Aromatic Amino Acids
Contains aromatic group (phenyl ring)
Participates in hydrophobic interactions
- Phenylalanine, Phe, F
- Tyrosine, Tyr, Y
- Typtophan, Trp, W
Tyrosine
Aromatic
Is like phenylalanine but has -OH, therefore making it more reactive
Basic amino acids
Positively charged
- Lysine, Lys, K
- Arginine, Arg, R
- Histidine, His, H
Charged, so found on the surface of proteins (interacts with water)
Lysine
Basic amino acid; has an amino group
Arginine
Basic amino acid; guanidinium group; side chains are positively charged at pH = 7 (pKa of the side chain is greater than 10)
Histidine
Typically considered a basic amino acid
Has ionizable group with pKa ~6
That means that it can be charged or uncharged depending on its location
Often found in active site of enzymes
Acidic amino acids
Negatively charged at pH = 7 (pKa < 4)
Contains carboxylic group
- Aspartate, Asp, D
- Glutamate, Glu, E
Charged, so found on the surface or proteins (interacts with water)
Polar amino acids
Not charged
Can form H-bonds (hydrophilic)
- Serine, Ser, S
- Threonine, Thr, T
- Cysteine, Cys, C
- Asparagine, Asn, N
- Glutamine, Glu, Q
Serine
Polar amino acid
Contains hydroxy group
Hydroxy group
R-OH functional group
Threonine
Polar amino acid
Contains hydroxy group
Cysteine
Polar amino acid
Contains sulfhydryl group (thiol group, -SH)
Can form disulfide bonds with another cysteine in the same chain or another. For example, insulin has 3 disulfide bridges.
Can form H-bonds, but they’re weak.
Asparagine
Polar amino acid
Derivative of aspartate
Contains carboxyamide instead of carboxyl
Glutamine
Polar amino acid
Contains carboxamide instead of carboxyl
pKa value of amino acids depend on ______
pKa value of amino acids depend on the environment
Primary structure
Linear sequence of amino acids linked by peptide bonds to form a protein
Peptide bond
linkage of alpha-carboxyl of one amino acid to the alpha-amino group of another amino acid, with the loss of water
Not energetically favourable to form, but once formed, it is stable (requires energy to be made)
Peptide bond has double bond characteristics because of resonance between the peptide bond and the carbonyl; as a result, it is planar
There is no free rotation about hte peptide bond
Because of steric hindrance, almost all peptide bonds are in trans-configuration (R-groups of opposite sides of the plane) except X-Proline (both cis and trans occur, but trans is preferred)
The peptide bond is conformationally restrained, but the bonds between N-Cα and Cα-C=O are single and free to rotate; thus, polypeptides are flexible
Polypeptide
A series of amino acids linked by peptide bonds
Has polarity; amino group is on the left and the free carboxylic group is on the right
Consist of repeated backbone with variable side chains
All carbonyl and amino groups in the backbone can H-bond (w/ the exception of proline); has an important role in 3D structure
Write the single letter code sequence of this polypeptide
SGYAL
Draw the polypeptide: SGYAL
Draw the formation of a peptide bond
Draw the formation of a disulfide bridge
Dihedral angle
The amount of rotation; the angle between planes through two sets of three atoms
Ranges from -180° to 180°
N-Cα is called Φ (phi)
Cα -C=O is called Ψ (psi)
N-C=O is the peptide bond… no free rotation
In reality, not all combinations of Φ and Ψ are allowed, because of steric hindrance that limits the number of structures a protein can adopt
Ramachandran plot
Shows the combinations of Φ and Ψ that are allowed
Areas of dark = favourable
Areas of light = borderline
Areas of white = not allowed
Each plot is for a particular amino acid; proline and glycine would have Ramachaundron plots that look very different from the other amino acids.
Proteins have a series of […] on what […] they can adopt. They can foten hold into […] […] structure in […] conditions.
Proteins have a series of limitations on what orientations they can adopt. They can often hold into a single structure in physiological conditions.
Why is knowing primary structure important?
- Determine 3D shape
- Understand function
- Understand dsease (ie. replacing a positiv AA with a negative AA is bad!)
- Understand evolutionary history
Secondary structure
The spatial arrangement of amino acid residues that are relatively close to each other in linear sequence of polypeptide chain (alpha helices, beta sheets)
Alpha helix
Polypeptide backbond forms the inner part of a right handed helix, with the side chans sticking outwards. It is stabilized by hydrogen bonds between NH and carbonyl (C=O) of the backbone
The C=O (i) forms H-bond with N-H (i+4) — he specifically said we need to know this
Has ideal dihedral angles of Φ = -60° and Ψ = -45°
All NH and C=O in the backbone are hydrogen bonded
Each aa in the a-helix increases the helix by 1.5Å (angstroms)
Proline/Glycine are the worst for alpha helix
R-groups in i+3 and i+4 are close to i
a-helix is right-handed; left-handed in possible but not stable due to steric hindrance
a-helical content vary in proteins (sometimes high, sometimes low)
Keratin consists of 2 intertwined helices
a-helix is shown as ribbons or rods in protein structures
Antiparallel beta sheet
Two or more B-strands associated as a stack of chains in an extended zigzag; form stabilized by interstrand H-bond
Each aa extends B-strand by 35Å
The N-H and C=O of residue “i” in one B-strand from H-bond to a single residue “j” in the other B-strand
Φ= -139° and Ψ= +135°
Parallel beta sheet
N-H of residue i of one strand forms H-bond with C=O of j in another strand, but C=O of i forms H-bond with N-H of j+2 residue in another strand
This makes strand shorter (3.25Å per residue)
Φ = -119° and Ψ= +113°
Draw where the hydrogen bonds would form within an a-helix.
Is this antiparallel or parallel?
Antiparallel
Is this antiparallel or parallel?
parallel
Draw the hydrogen bonds on this beta sheet and state the Å
Draw the hydrogen bonds and label the Å
How are beta sheets depicted?
broad arrows pointing to C-terminus
Turns and loops
Peptide chains reverse direction
Accomplished by B-turn (common) or by larger loops (no common structure)
What is this?
A beta barrel – a transmembrane protein
Tertiary structure
The spatial arrangement of aa residues that are far apart from each other in linear sequence as well as the pattern of disulfide bonds
Unique for every protein
What is this? Describe it.
Myoglobin
153 amino acids, relatively small
Largely a-helices (70%)
Globular
Few voids (unorganized chain)
In red = heme (protoporphyrin ring) = where oxygen binds
Surface contour
Yellow = hydrophobic amino acids; mostly in protein core
Polar amino acids on the outside
What is this? Describe it.
Troponin C
Ca2+ binding proteins
2 domains - parts of protein with defined function
Quarternary structure
The spatial arrangement of multiple subunits (polypeptides) and their interaction
Some proteins are composed of more than one polypeptide chain (multimers) in order to function
What is this? Describe it.
Hemoglobin; Hb
O2 transporter in blood of mammals
4 subunits: 2 alpha + 2 beta
Hb cannot function unless it forms tetramer (4 peptides)
What is this? Describe it.
Minute virus of mice
9 VP1 + 51 VP2 = viral capside (quarternary structure)
Large enough to fit DNA
Three examples of quarternary structures
- Proteasome
- Spliceosome
- Ribosomes (+ RNA)
In nature, polypeptides […] folds into one structure in seconds.
USUALLY, NOT ALWAYS
How do we predict the final 3D conformation?
We cannot predict the final 3D conformation using primary structure. RIP.
What do we know about protein folding, if we can’t predict the final 3D conformation then?!
What we do know:
Folding is driven by thermodyanmics: finding the most stable complex (most negative ∆G), but the difference between folded and unfolded protein is small (~20-60 kJ/mole)
Driven mostly by entropy (ie. the hydrophobic side chains are going to be exluded from water in the core, while polar amino acids are at the surface)
In order to fold, hydrogen bonds must form
Unpaired charged or polar groups will destabilize structures.
What can we do?
Though we cannot predict final 3D structures, we can predict secondary structures.
Certain amino acid residues are more likely to form a-helix or B-sheets
A, L, K, M form stable a-helix
Proline and glycine destabilize a-helix
P doesn’t have H and it is rigid (steric hindrance)
G is too flexible
However, even peptides with the same sequence can adopt different secondary structure in different proteins
Folding is usually an all-or-none process (either folded or misfolded/unfolded)
Folding is cooperative (if one portion of the protein folds, it will influence how another portion of the same protein folds)
We usually visualize protein folding as a free energy funnel
Normal tertiary structure = native structure = functional protein
In a protein’s unfolded state, there are many possible structures with high free enrgy, but as a series of folding happens, free energy decreases with every structure formed. The number of possible conformations decrease until you reach the native (folded) state
Note: Not all proteins have one single conformation
Some might only form a final structure when bound to a substrate or regulator; some exist in equilibrium between two different structures
What are the three ways to determine the final 3D structure of proteins?
Cryoelectron microscope: uses a beam of electrons to image many, many native proteins
X-ray crystallography: measure e- density (ie. myoglobin)
NMR (nuclear magnetic resonance): measures the location of nuclei
Posttranslational modifications
Modifications to proteins after it has been synthesized
- Phosphorylation - adding phosphate to amino acids with -OH (ie. Ser, Tyr, Thr); ie. signal transduction to activate or deactivate proteins
- Glycosylation - addition of sugars to a residue (usually Asn and Ser); ie. cell recognition (immune system); ie. glycoprotein
- Hydroxylation - usually protein (hydroxyproline); ie. collagen - triple helix
- Carboxylation - addition of a carboxyl group; glutamate (ie. blood and clotting)
- Acetylation - addition of acetyl group; lysine - regulation of gene expression (epigenetics)
- Proteins can be trimmed (ie. trypsinogen –> trypsin)
Label these posttranslational modifications
- Hydroxylation
- Carboxylation
- Glycosylation
- Phosphorylation
Enzyme
Biological macromolecule that acts as a catalyst for biochemical rxns. Usually proteins.
Catalyst
Chemical that increases the rate of rxn without being consumed
Kcat
of molecules of substrate converted to product per molecule of enzyme per second
Specificity
Enzymes are very specific; they will catalyze only one specific reaction or a set of rxns
Specificity is based on a series of weak interactions between substrate and enzyme, especially in the active site
The shape of the enzyme determines specificity and function
Trypsin
Digestive enzyme
Cleaves peptide bond on the carboxyl side of Lys or Arg
Papain
Cleaves any peptide bond
Thrombin
Involved in blood clotting
Cleaves Arg (or Gly) peptide bonds
What digestive enzyme is this?
Trypsin
What digestive enzyme is this?
Thrombin
Active site
Region of an enzyme that binds the substrate. It contains the residues that directly participate in the rxn. The nonaxtive site residues are important for structure of the protein or regulation of enzyme (ie. phosphorylation/inhibitors)
Characteristics of an active site
- They are clefts in the enzyme made up by residues from all over the primary structure
- Takes up a small volume of an enzyme
- Water is usually manipulated or excluded from an active site
– This changes the behaviour of the resiudes
- Substrate is bound by weak interactions in active site
- There is a partial complimentarity between substrate and active site
Models for how substrates may bind in the active site of an enzyme
- Lock and Key
- Active site is complimentary to match to a substrate - Induced Fit
- Binding of a substrate causes the active site to assume a matching shape
Cofactors
Enzymes may require cofactors in order to function
Cofactor: inorganic ion or small organic compound required for enzyme activity
Cofactor vs Coenzyme
Cofactor - only inorganic ions (ie. Mg2+)
Coenzyme - organic compounds (ie. FAD)
Prosthetic group
Cofactor that is tightly bound to an enzyme (heme in myoglobin)
Apoenzyme
Enzyme that requires a coenzyme but doesn’t have the cofactor (no function)
Holoenzyme
Enzyme that requires a cofactor and has the cofactor (functional)
Ni2+
This is a cofactor that is found in tap water and needed for DNA synthesis
Enzymes Thermodynamics
- Enzymes do not alter ∆G; they obey laws of thermodynamics; do not change how spontaneous rxn is
- Enzymes do not alter the final equilibrium of products to reactants
- Enzymes do speed up rxns; enzymes accelerate rxns by decreasing the activation energy (∆G‡) by facilitating the formation of the transition state (X‡)
The energy needed to get to X‡ is known as activation energy (∆G‡)
∆G‡s→p = Gx‡ - Gs → forward rxn
∆G‡p→s = Gx‡ - Gp → reverse rxn
Only a fraction of substrate (s) will have enough energy to form X‡
The activation energy cotnrols the rate (rate limiting step)
Activation energy is not a part of the ∆G of the rxn
∆G‡cat < ∆G‡uncat
This is how enzymes work:
S+E ⇌ ES ⇌ EP ⇌ E + P
Enzymes will interact with transition state such that the activation energy is lowered: the rxn will speed up as a greater fraction of S has energy to react (to convert to X‡)
Where does the energy to lower ∆G‡ come from?
It comes from the enzyme binding and stabilizing the transition state in the active site. Active site is most complimentary to X‡
Binding energy
∆GB = binding energy
Defined as the energy derived from the non-covalent interactions between the enzyme and substrate
How can we measure the rate of rxn for
A → P
We can measure the rate (or velocity) of this rxn as eithr disappearance of substrate A over time or the appearance of product (P) over time.
V = -∆A/∆t = ∆P/∆t (usually expressed as moles/unit of time)
If we measure the disappearance of A, then we can express the rate as directly related to [A]
V = k[A] k - rate constant (not an equilibrium constant!)
k = proportionality constant that relates to [S] at a specific temperature.
This is known as first order rxn and k has units of sec-1 or min-1
Second order kinetics
2A → P
A + B → P
2A → P where v = k[A]2
A + B → P where v = k[A][B]
These 2nd order rxns usually have k as units of M-1sec-2 or M-1min-1 or moles-1sec-1
Michaelis-Menten Kinetics
Series of tubes with fixed amount of enzyme, mixed with different amount of substrate. Then they measured the amount of product formed over time.
Draw a graph of free energy G vs reaction coordinate for an exergonic reaction. Label reactants, products, activation barrier/transition state (‡), ∆G‡cat, ∆G‡uncat and ∆G
Draw a graph of free energy G vs reaction coordinate. Label S (ground state), Transition State (‡), P (ground state), ∆G‡S→P, ∆G‡P→S, ∆G’°
Draw a graph of free energy G vs reaction coordinate. Label S, P, ES, EP, transition states (‡), ∆G‡uncat, ∆G‡cat, ∆Grxn
Who are these people?
Leoner Michaelis
Maud Menten
Assumptions about enzyme kinetics
- We only examine early times in the rxn when [P] is low. We can ignore reverse rxn.
E + S ⇌ ES → P
- [S] >> [E], we can assume that [ES] doesn’t affect [S]
- A steady state exists such that the rate of [ES] formation = rate of [ES] consumption
Caculating the initial velocity
We can calculate the initial velocty, V0 (initial rate, μM/time)
Based on an earlier assumption, we can describe how initial velocity depends on [S]
V0 = Vmax ([S]/([S}+Km))
[S] = concentration of substrate
V0 = initial velocity
Vmax = maximum velocity of rxn, when all the active sites are saturated with subtrate
KM = Michaelis constant; [substrate] at which the enzyme catalyzed rxn proceeds at 1/2 Vmax rate. It is a rate constant.
Michaelis constant
KM = K-1 + K2 / K1
When [S] << KM => V0 = Vmax ([S]/KM)
[S] = KM => V0 = Vmax (KM/2KM) = 1/2 Vmax
[S] >> KM => V0 = Vmax ([S]/[S]) = Vmax
KM is an important characteristic of enzymes; it’s a rough estimate of affinity of the enzyme to its subtrate
KM high = affinity is low
KM low = affinity is high
It depends on the type of enzyme, pH, temperature, ionic strength
Vmax
Maximum rate of enzyme
It will change with [enzyme]
What is this called?
Lineweaver-Burk plot
What is the x-intercept of the Linweaver-Burk plot?
-1/KM
What is the y-intercept of the Lineweaver-Burk plot?
1/Vmax
Do all enzymes follow Michaelis-Menten kinetics?
No.
List the 4 types of enzyme inhibition
- Irreversible inhibition
- Competitive inhibition
- Non-competitive inhibition
- Uncompetitive inhibition
Can KM and Vmax be used for comparing enzymes?
No, because they change depending on the concentration of enzyme used.
How can we compare enzymes, if not with KM or Vmax ?
We can use Kcat !
Kcat
The minimum amount of subtrate an enzyme can convert into product in a given time (min-1 or sec-1)
Kcat = Vmax/[E]T
[E]T = enzyme total concentration = [E] * # of active sites
Higher number = better / more efficient enzyme
Specificity constant / Catalytic efficiency constant
Kcat / KM
sec-1M-1
Label the axes, the slope, and the intersepts
Irreversible inhibitor
AKA suicide inhibitor
Inhibitor stays boundt o enzyme for long periods (often forever). Usually covalently attached in active site => inhibitor blocks active site
Competitive inhibitor
- inhibitor binds to the active site and competes with the substrate for it
- only one can bind at a time
- Vmax doesn’t change but amount of subtrate needed to reach Vmax – as well as half of Vmax – is increased (KM increases)
- Adding inhibitor increases KM but Vmax remains unchanged
- Can be overcome by adding more subtrate
- In a double-reciprocal plot, Y-intercept stays the same; X-intercept increases (KM increases, because -1/KMi > -1/KM => KMi > KM
Non-competitive inhibitor
- Inhibitor binds at a site other than the active site
- Inhibitor doesn’t block active site (ie. substrate can bind to active site, but products are not formed)
- can’t compete by adding more substrate
- KM doesn’t change, but Vmax decreases
- In effect, you are lowering the number of functional enzymes
- In double-reciprocal plot: x-intecept is the same, but y-intercept is different (increases)
Uncompetitive inhibitor
- Inhibitor only binds to ES, forming ESI complex
- Like non-competitive inhibition, this results in lower Vmax (lowering # of functional enzymes)
- But it also decreases KM because as ES → ESI => [ES] ↓ ! This promotes increased substrate binding to enzyme, lowering KM
- In the double reciprocal plot: both X and Y-intercepts change => parallel lines
– x-intercept decreases (moves left) and y-intercept increases (moves up)
What type of inhibition is this?
Uncompetitive
What type of inhibition is this?
Noncompetitive
What type of inhibition is this?
Competitive
Draw the line corresponding to competitive inhibition on this plot.
Draw the line corresponding to noncompetitive inhibition on this plot.
Draw the line corresponding to uncompetitive inhibition on this plot.
