CC 1 A: Structure & function of proteins and their constituent amino acids Flashcards
Amino Acid classifications & protein structural elements are covered. Special emphasis is placed on enzyme catalysis, including mechanistic considerations, kinetics, models of enzyme-substrate interaction, and regulation.
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YOU GOT THIS <3
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KEEP GOING!!
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Amino Acids
Absolute configuration at the α position
A
- The alpha carbon IN EVERY amino acid is a chiral center EXCEPT in glycine (it is achiral, since the R group is an H)
- EVERY AA has S configuration EXCEPT FOR cysteine (R configuration)
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Q
Amino Acids as dipolar ions
At low pH, amino acid = ?
At high pH, amino acid = ?
At pH = pI, amino acid = ?
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At low pH, amino acid = cationic
At high pH, amino acid = anionic
At pH = pI, amino acid = zwitterionic (neutral)
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Classify Amino Acids
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- Acidic or Basic
- Acidic: Aspartic Acid (Asp, D); Glutamic Acid (Glu, E)
- Basic: Lysine (Lys, K); Arginine (Arg, R); Histidine (His, H)
- Hydrophobic or hydrophilic:
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Hydrophilic: If the R group contains acids, bases, amines or alcohols
- Arginine (Arg, R), Lysine (Lys, K), Aspartic Acid (Asp, D), Glutamic Acid (Glu, E), Glutamine (Gln, Q), Asparagine (Asn, N), Histidine (His, H), Serine (Ser, S), Threonine (Thr, T), Tyrosine (Tyr, Y), Cysteine (Cys, C), Tryptophan (Trp, W)
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Hydrophobic: If the R group DOES NOT contain what is listed above ^^
- Alanine (Ala, A), Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met,M), Phenylalanine (Phe, F), Valine (Val, V), Proline (Pro, P), Glycine (Gly, G)
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Hydrophilic: If the R group contains acids, bases, amines or alcohols
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Amino Acids Reactions
Sulfur Linakage for cysteine and cystine:
A
- Cysteine = amino acid with the thiol R group
- Cystine = 2 cysteines that have formed a disulfide bond
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Amino Acid Reactions
Peptide Linakge: polypeptides & proteins
What links aa chains together?
A
- Peptide bonds link amino acid chains together
- Peptide bonds are formed by the nucleophilic addition-elimination (condensation, dehydration rxn) reaction between the carboxyl group of one amino acid and the amino group of another amino acid
- The nucleophilic amino group attacking an electrophilic carbonyl
- The bond when formed has a lot of resonance delocalization (partial double bond character all over the place!)
- Makes the bond very rigid/planar
- However, this is still free rotation around the ALPHA CARBON
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Amino Acids
Hydrolysis
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- The process of breaking the peptide bond
- Done by either acid/base hydrolysis (nonspecific) or with the help of proteolytic enzymes (specific)
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Primary Structure of Proteins
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- Linear sequence of amino acids
- determined by the peptide bond linking each aa
- Covalent (peptide) bonds
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Secondary Structure of Proteins
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- Local structure, stabilized by hydrogen bonding
- α-helices – hydrogen bonds run up and down, stabilizing the structure
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β-pleated sheets – stabilized by hydrogen bonds connecting the sheets
- Antiparallel vs. Parallel configurations
- The way the linear sequence folds on itself
- Determined by the backbone interactions (primarily hydrogen bonds)
- Hydrogen bonds between backbone atoms
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Tertiary Structure of Proteins
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- 3-D structure stabilized by hydrophobic interactions, acid-base interactions (sallt bridges), hydrogen bonding, and disulfide bonds
- Depends on distant group interaction
- stabilized by hydrogen bonds, van der waals, hydrophobic packing, disulfide bridge formation
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Disulfide bond formation happens on the exterior of the cell (covalent bond of two cysteines)
- Extracellular space prefers the formation of disulfide bonds (the oxidizing environment)
- Hydrophobic interactions & polar interactions between side chains
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Quantnary structure of proteins
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- Interactions between subunits (multiple polypeptides)
- Hydrophobic interactions and ionic bonds between side chains (i.e. cysteine side chains making disulfide bonds)
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Conformational Stability
Denaturing & Folding
What dorce helps stabilized the protein?
A
- Primary Structure = determined by peptide bonds
- Secondary Structure = determined by backbone interactions (hydrogen bonds)
- Tertiary Structure = determined by distant interactions between groups (van der Waals, hydrophobic packing, disulfide, hydrogen bonding)
- Quaternary Structure = determined by same bonds from tertiary structure
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Protein is ONLY FUNCTIONAL when in the proper conformation
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**A force that helps stabilize the protein is the solvation shell
- Solvation shell = layer of solvent surrounding the protein (can be the water solvent interaction with polar AAs, etc.)
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**A force that helps stabilize the protein is the solvation shell
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Denaturation: when a protein losses active conformation & becomes inactive
- occurs by changing pH, temp, chemicals or even enzymes
- If you denature by heating, you destroy all the stuctures of the protein except the primary structure (primary structure is conserved)
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Conformational Stability
Hydrophobic interactions/Solvation Layer (entropy)
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- the hydrophobic regions of the protein aggregate, which releases the water from cages
- this increases the entropy of water, which is the major thermodynamically favorable component of protein folding
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Solvation Layer & Entropy
ORDER=?
DISORDER=?
WHAT HAPPENS TO THE PROTEIN WHEN IT FOLDS? WHAT HAPPENS TO THE WATER MOLECULES THAT ARE SURROUDING THE PROTEIN WHEN IT FOLDS?
A
- The polarity and charge of amino acid residues on the surface of a protein affect the order of the surrounding water molecules, as measured by entropy, ΔS.
- Entropy is a measure of the disorder within a system.
- Increased order is=negative entropy change
- Increased disorder = to a positive entropy change.
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The water molecules surrounding folded proteins have higher entropy than those surrounding unfolded proteins because the hydrophobic molecules on the surface of unfolded proteins force water to form a rigid solvation layer.
- protein becomes more ordered when it folds BUT the water molecules surrounding it becomes more DISORDERED
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Seperation Techniques
Isoelectric Point (pI)
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- pI is determined by averaging the pKa values that refer to the protonation & deprotonation of the zwitterion
- Isoelectric focusing: gel electrophoresis method that seperates proteins on basis of their relative contents of acidic and basic residues (gel with pH gradient is used)
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Seperation Techniques
Electrophoresis
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- positively charged anode at bottom, negatively charged cathode at the top
- larger molecules will have harder time moving, thus seperation created by size with the smallest molecules towards the bottom
- Native Page: retains structure of preotein; SDS-Page: break into subunits
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Non-Enzymatic Protein Function
Binding
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- Bind various biomolecules – bind specifically and tightly
- Receptors/Ion channels in the membrane:
- Receptors bind or receive signaling molecules (ligand) which makes a chemical response (i.e. insulin receptor)
- Ion channels can allow ions to enter/exit the cell
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Non-Enzymatic Protein Function
Immune System
What are antibodies?
What are antigens?
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- Antibodies: protein components of the adaptive immune system whose main function is to find foreign antigens and target them for destruction
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Antigens: the ligand for antibodies
- Antigens can be thought be thought of as little red flags for the immune system letting us know, “hey, that’s not supposed to be there?”
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Non-enzymatic protein function
Motors
Transport example
Mysosin/Kinesin/Dynein
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- Transport: e.g. hemoglobin (at high concentration of ligand=have high affinity, at low concentration of ligand=have low affinity
- Myosin=responsible for forces exerted by contracting muscles
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Kinesin/Dynein=motor proteins responsible for intracellular transport
- Dynein=plays a role in the motility of cilia
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Activation Energy i the uncatalyzed and catalyzed reactions, draw graphs:
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- The activation energy Ea is the minimum energy required for reactants to reach the transition state and initiate a reaction (given by Ea = E‡ − Ereactants).
- Uncatalyzed reactions have greater Ea than catalyzed reactions, and therefore are slower.
- Catalysts allow the reaction to proceed at a faster rate by lowering the activation energy.
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- Enzyme Energy parameters & the effect on reactions:
- Deleterious mutations involving the enzyme’s active site are likely to:
A
- Enzymes do not alter the free energy change ΔG or the equilibrium constant Keq of a reaction
- but do decrease the time taken for the reaction to reach equilibrium by increasing the reaction rate.
- Deleterious mutations involving the enzyme’s active site are likely to alter the enzyme’s affinity for its substrate (increase Kd) and interfere with enzymatic function (decreased reaction rate).
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Q
Transferase
A
- Move a functional group from one molecule to another
- A + BX –> AX + B
- Example: phosohorylases are a type of transferase that breaks bonds by adding a phosphate, as in glycogen degradtion
- cannot catalyze redox reactions
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Oxidoreductase
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- Catalyze oxidation-reduction reactions that involve the transfer of electrons
- Oxidase = oxidizing or taking away electrons from a molecule (OIL)
- Reductase = reducing or giving electrons to a molecule (REG)
- A + B: <—-> A: + B
- EXAMPLE: cleavage of protein disulfide bonds by eznme thioredoxin (catalyzes the transfer of electrons to break the bond)
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Isomerase
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- Interconversion of isomers, including both constitutional and stereoisomers
- rearange functional groups within a molecule but do not catalyze redox reactions
- A –> B
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Hydrolase
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- cleavage with the additon of water, break peptide bonds by the addition of water
- A + H2O –> B + C
- Example: proteases break peptide bonds by the addition of water
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Substrates & Enzyme Specifity
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- Enzyme-substrate specificity derives from structural interactions
- Enzymes can be specific enough to determine between stereoisomers
- some enzymes have high specifity for their substrates
- these enzymes catalyze only reactions with a particular substrate (has high reaction rates for substrates of interest) and do not act on other molecules with similar, but not identical, structure
- Example: the α-amylase enzyme cleaves the glycosidic bonds in starch but not cellulose, even though the two molecules differ only in the orientation of their glycosidic bonds.
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Induced-fit Model
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Initial Binding: when the substrate first binds to the enzyme (not perfect)
- Forces holding the two together are strong, but not at the maximum strength yet
- Enzyme and substrate thus mold their shape to bind together super tightly
- called the induced fit because both the enzyme & substrate have changed their shape a little so they can bind together really tightly (catalyzing reaction at full force)
- example: when a protein changes confirmation from 3 to 4, this requires energy so it would be the induced fit
- called the induced fit because both the enzyme & substrate have changed their shape a little so they can bind together really tightly (catalyzing reaction at full force)
- Binding between reactant and enzyme STRONGEST at the transition state
lock and key theory proposes that an enzyme’s active site is already in the proper structural conformation to allow a substrate to bind readily and form an active enzyme-substrate (ES) complex. This theory proposes that no conformational changes are necessary for catalysis to occur.
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Mechanism of Catalysis
- Cofactors
- Coenzymes
- Water-Soluble Vitamins
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Cofactors: directly involved in the enzyme’s catalytic mechanism (might be stabilizing the substrates, or helping the reaction to convert substrates from one from to another) (e.g. Mg+)
- inorganic
- enzymes that require cofactors are called apoenzyme when the cofactor is absent; where as those containing cofactors are known as holoenzymes
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Coenzymes: organic carrier molecules (i.e. NADH, CoA, THF, FADH2)
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Water-Soluble Vitamins: need to obtain from the diet
- Vitamins–>organic cofactors & coenzymes
- e.g. vitamins B3 is a precursor for NAD
- e.g. Vitamin B5 is a precursor for CoA
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Water-Soluble Vitamins: need to obtain from the diet
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Q
Michaelis-Menton (MM)
What is Vmax?
MM equation calculates?
High enzyme substrate affinity means, HIGH or LOW Km?
Vice versa?
A
- Vmax is defined for a specific enzyme concentration (adding more enzyme will increase the Vmax)
- Michaelis-Menten equation calculates the rate of reaction using Vmax, the substrate concentration [S], and the Michaelis constant Km. Km = the [S] required to reach 1/2Vmax
- V=vmax [S] / Km + [S]
- As substrate concentration increases, the reaction rate also increases until a maximum value is reached
- At 1⁄2 Vmax, [S] = Km
- Km does not fluctuate with changes in [enzyme] and is indicative of enzyme-substrate affinity
- Enzymes with high enzyme-substrate affinity will reach 1/2Vmax at a lower substrate concentration (Lower Km)
- Lower enzyme-substrate affinities will result in needing a higher substrate concentration to reach 1/2Vmax (Higher Km)
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Hill Coefficient:
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- indicates cooperativity
- results in a SIGMOIDAL curve
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Competitive Inhibition
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- E (inhibitor binds to E here to make EI) + S <—-> ES <—-> E+P
- Blocks the enzyme and makes it unable to react with substrate to form product
- Inhibitor competes with substrate for space on the enzyme
- Binds: Active Site
- Impact on Km: Increases
- Impact on Vmax: No Change
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Uncompetitive Inhibition
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- E + S <—-> ES (inhibitor binds to the ES here to make ESI) <—-> E + P
- Molecule that binds only to the enzyme-substrate complex, rendering it catalytically inactive
- Binds: Allosteric Site
- Impact on Km: Decreases
- Impact on Vmax: Decreases
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Non-Competitve Inhibition:
A
- Prevents the enzyme from turning substrate into product
- Binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site regardless of whether a substrate is already bound
- Binds: Allosteric Site
- Impact on Km: No Change
- Impact on Vmax: Decreases
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Irreversible Inhibitor & Reversible inhibitors
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Irreversible inhibitors form covalent bonds with enzymes and become more potent given sufficient time to react.
- react with nucleaophilic side chains to form the covalent bonds
- Preincubation with inhibitor provides more time for covalent linkages to occur; therefore, it can increase the level of inhibition because more enzymes will be inactivated.
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Reversible inhibitors quickly form noncovalent bonds with target enzymes and do not require much time to achieve their full effect.
- tend to have the same effect like irreversible, whether or not they are preincubated with an enzyme
