Proteins & Enzymes Flashcards
enzymes
Enzymes are biological catalysts - they increase reaction rates without being used up
Most enzymes are globular proteins.
Note: some RNA (ribozymes and ribosomal RNA) also catalyse reactions. A lot of factors that can bind to an enzyme to switch it on or off.
biocatalysts over inorganic catalysts
Enzymes have:
- Greater reaction specificity: avoids side products
- Milder reaction conditions conducive to conditions in cells: eg. pH ~7, 37C
- Higher reaction rates - biologically useful timeframe
- Regulation: control of biological pathways: Phosphorylate to switch on or off, add side chains, allows enzyme regulation
Metabolites have many potential pathways of decomposition - Enzymes makes the desired reaction most favourable (eg on right)
enzyme substrate selectivity (specific)
complex drives selectivity
different stereochemical arrangement prevents binding of enzyme to the stereoisomer of a molecule (no binding).
- Analogue: can bind but no reaction. Because particular analogue has enough complementarity to allow binding, but enzyme cannot function because not right molecule, not a perfect fit.
- Binding but no reaction: becomes an inhibitor
enzymatic catalysis
enzymes do not affect equilibrium (Keq) therefore cannot effect ΔG
slow reactions face significant activation Barries ΔG‡ that must be surmounted during the reaction
- Enzymes increase reaction rates (k) by decreasing ΔG‡.
- ΔG‡ = Gtransition state – Greactants
reaction coordinate diagram
the free energy of the system is plotted against the progress of the reaction S –> P.
graph describes energy changes during the reaction.
The activation energies, ∆G‡, for the S –> transition state are indicated.
∆G ’ ° is the overall standard free-energy change in the direction S –> P.
Negative ∆G ’ ° mean favourable – but significant activation energy can prevent it from progressing spontaneously.
how to lower ∆G‡
Enzymes organize reactive groups into close proximity and proper orientation.
Uncatalyzed reactions may be entropically (energetically/thermodynamically) unfavorable.
Catalyzed reactions:
o The enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex.
o The entropy cost is paid during binding.
o Rigid reactant complex –> transition state conversion is entropically neutral. (reaction now favourable)
- Enzymes bind transition states best.
catalytic mechanisms (3)
Enzymes may use one or a combination of the following:
- acid-base catalysis: give and take protons
- covalent catalysis: change reaction paths
- metal ion catalysis: use redox cofactors, pKa shifters
covalent catalysis
a transient covalent bond between the enzyme and the substrate. changes the reaction pathway via breaking bond - uncatalysed A - B --> A + B - catalysed (X is catalyst) A - B + X: --> A - X + B --> A + X : + B
metal ion catalysis
Involves a metal ion bound to the enzyme, Interacts with substrate to facilitate binding
- stabilizes negative charges
- Participates in oxidation reactions
enzyme kinetics
kinetics: study of the rate at which compounds react.
rate of enzymatic reaction is affected by: enzyme, substrate, effectors, temperature (up to a point, then denaturation)
determination of kinetic parameters
Michaelis-Menten and lineweaver-burke
A non-linear Michaelis-Menten plot should be used to calculate parameters Km and Vmax.
At a certain point: RR plateaus (fixed amount of enzyme in tube, and only 1 binding site so once conc of substrate exceeded, enzyme is used up)
o Vmax: max velocity for reaction, never reached (hypothetical line)
o ½ Vmax = Km. Km constant for r’n
Lineweaver Burke Plot is just a mathematical way of rearranging the data to create a plot with a straight line.
o y axis = 1/Vo (1/initial velocity)
o x axis = 1/[S] (1/substrate concentration)
o small Km = fast RR (inverse proprotional)
forms of enzyme inhibition
Inhibitors are compounds that decrease an enzyme’s activity.
Irreversible inhibitors (inactivators) react with the enzyme.
o 1 inhibitor molecule can permanently shut off 1 enzyme molecule.
o They are often powerful toxins but also may be used as drugs( Eg snake venom, cyanide etc)
Reversible inhibitors can bind and dissociate from enzyme.
o They are often structural analogs of substrates or products.
o They are often used as drugs to slow down a specific enzyme.
- Reversible inhibitor can bind to:
o the free enzyme and prevent the binding of the substrate.
o the enzyme-substrate complex and prevent the reaction.
competitive inhibition
competes with substrate for binding - binds to active site. does not affect catalysis. No change in Vmax, increase in Km.
Lineweaver-Burk: lines intersect at the y-axis (1/Vmax) –> Vmax unchanged (1/Vmax). Increasing Km
uncompetitive inhibition
only binds to ES complex, does not affect substrate binding, inhibits catalytic function. - Decrease in Vmax; apparent decrease in Km. No change in Km/Vmax
Lineweaver-Burk: lines are parallel.
mixed inhibition
Binds enzyme with or without substrate, binds to regulatory site, inhibits both substrate binding and catalysis
Decrease in Vmax; apparent change in Km
Lineweaver-Burk: lines intersect left from the y-axis. Noncompetitive inhibitors are mixed inhibitors such that there is no change in Km.
enzymes activity regulation: pos and neg
Regulation can be:
1. Non-covalent modification (allosteric)
o Regulator interacts non-covalently with enzyme
o Can be positive or negative (activate or inhibit)
- Allosteric regulators are:
o generally small chemicals
o can be positive, or improve enzymatic catalysis.
o can be negative, or reduce enzymatic catalysis.
2. Covalent modification o Irreversible o Reversible o Can be positive or negative (activate or inhibit) o Eg phosphorylation
**some enzymes use multiple types of regulation, important in metabolism
protein structure
protein molecules adopt a specific 3D conformation. This structure is called the native fold, and allows protein to fulfil its specific function.
native fold
has a large number of favourite interactions within the protein. native fold = form/fold in which it is functional.
There is an entropy cost to folding the protein into one specific native fold.
primary structure
the peptide bond
sequence of AA joined covalently by a peptide bond, structure of the protein is partially dictated by the properties of the peptide bond
resonance in peptide bond: each peptide bond has double bond character due to resonance and therefore cannot rotate.
resonance causes peptide bonds to be less reactive (very strong), rigid and planar (not free to rotate) and favoured trans configuration has dipole (=O δ- NH δ+)
secondary structure
local spatial arrangement of polypeptide backbone
2 common regular arrangements
α helix: stabilised by H bonds between nearby residues (residue: specific monomer within the polymeric chain of a polysaccharide, protein or nucleic acid), creates helical structure
β sheet: stabilised by H bonds between adjacent segments that may not be nearby, create pleated sheet like structure, sheets can run
- parallel: H bonds between strands are bent (weaker)
- antiparallel: H bonds between strands are linear (stronger)
irregular arrangement of polypeptide chain is called the random coil
tertiary structure
overall spatial arrangement of atoms in a protein, stabilized by numerous weak interactions between amino acid side chains
largely hydrophobic and polar interactions, can be stabilized by disulfide bonds
Interacting amino acids are not necessarily next to each other in the primary sequence.
Two major classes: fibrous and globular (water or lipid soluble)
Can be: hydrogen bonds, ionic bonds, van der Waals forces, cross-linking by disulphide bonds.
quaternary structure
formed by the assembly of individual properties into a larger functional cluster
protein stability and folding
factors that denature proteins
protein’s function depends on its 3D structure. loss of structural integrity with accompanying loss of activity is called denaturation.
Factors:
heat or cold, pH extremes, organic solvents, chaotropic agents (urea and guanidinium HCl), reducing agents (2ME)
Formation of covalent disulfide bonds: help stabilize between 2 polypeptide chains. Reducing agents break disulfide bonds therefore causing denaturation of the proteins
Function of globular proteins
Reversible binding of ligands is essential.
o Ligand = other molecules that bind specifically to a protein
o Require absolute specificity of ligands and binding sites
o Ligand binding is often coupled to conformational changes, sometimes quite dramatically (induced fit).
o In multi-subunit proteins, conformational changes in one subunit can affect the others (co-operativity).
o Interactions can be regulated.
Illustrated by:
o hemoglobin, antibodies, and muscle proteins
o Hb: O2 binds to one subunit allows easier access for other O2 molecules to bind to other sub units
types of globular proteins
Storage of ions and molecules: myoglobin, ferritin
Transport of ions and molecules: hemoglobin, serotonin transporter
Defense against pathogens: antibodies, cytokines
Muscle contraction: actin, myosin Biological catalysis (ENZYMES): chymotrypsin, lysozyme
interactions with other molecules
reversible, transient process of chemical eqm A + B AB
A molecule that binds to a protein is called a ligand, typically a small molecule.
region in protein where ligand binds = binding site. binding occurs via non-covalent interaction, dictate protein structure, allows interactions to be transient (can bind and then be released)
binding specificity (induced fit)
ligand binds via same non-covalent interactions that dictate protein structure.
- hydrophobic, disperion, H bonds, ionic
indued fit: Conformational changes may occur upon ligand binding, allows for tighter binding of the ligand and for high affinity for different ligands.
- both the ligand and protein can change their conformations
protein biological functions
Catalysis: enolase (in the glycolytic pathway), DNA polymerase (in DNA replication)
Transport: hemoglobin (transports O2 in the blood). lactose permease (transports lactose across the cell membrane)
Structure: collagen (connective tissue), keratin (hair, nails, feathers, horns)
Motion: myosin (muscle tissue), actin (muscle tissue, cell motility)
amino acid function and properties
building blocks of protein.
properties:
capacity to polymerise, acid-base properties, varied physical properties, varied chemical functionality
general AA structure
all AA (except proline) have: an acidic carboxyl group connected to the α carbon
a basic amino group connected to the α carbon
an α hydrogen connected to the α carbon
The fourth substituent (R) is unique in glycine, the simplest amino acid. The fourth substituent is also hydrogen.
all AA are chiral (except glycine)
AA classification
5 different groups depending on R substituents
- non polar, aliphatic R groups
- aromatic R groups
- polar, uncharged R groups
- positively charged R groups
- negatively charged R groups
AA ionisation
AA contain at least two ionisable protons, each have own pKa.
COOH: acidic pKa, protonated at a low pH
NH4+: basic pKa, proontaed until high pH achieved
low pH: AA in pos charged from - cation (COOH and NH4+ protonated)
high pH: AA in neg charged form - anion (COOH and NH4+ de-protonated).
Between the pKa for each group, the amino acid exists in a zwitterion form, in which a single molecule has both a positive and negative charge (pI).
AA as a buffer
AA with uncharged side chains, eg glycine have two pKa values,
As buffers prevent change in pH close to the pKa, glycine can act as a buffer in two pH ranges, because each AA have 2 pKas
AA charge (pI)
Amino Acids Carry a Net Charge of Zero at a Specific pH called the Isoelectric point (pI)
Zwitterions predominate at pH values between the pKa values of the amino (pK1) and carboxyl groups (pK2).
pH at which the Zwitterion form of the amino acids with neutral side chains exists = the isoelectric point pI.
AA polymerisation to form peptides
Peptides are small condensation products (monomers/monomeric unit) of amino acids
Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced.
At physiological pH, the reaction shown here does not occur to any appreciable extent.
Peptide bonds strongest and hard to break, and most stable
peptide functions
Hormones and pheromones
o insulin (think sugar metabolism)
o oxytocin (think childbirth)
o sex-peptide (think fruit fly mating)
Neuropeptides
o substance P (pain mediator)
Antibiotics
o polymyxin B (for Gram – bacteria)
o bacitracin (for Gram + bacteria)
Protection, e.g., toxins o amanitin (mushrooms) o conotoxin (cone snails) o chlorotoxin (scorpions)
peptide composition
Polypeptides (covalently linked α amino acids) + possibly: cofactors: functional non-amino acid component, metal ions or organic molecules
coenzymes: organic cofactors, NAD+ in lactate dehydrogenase
prosthetic groups: covalently attached cofactors, haem in myoglobin
other modifications (post-translational modifications)
Km definition
indicates the affinity of the enzyme for its substrate
low Km = high affinity
K, is the concentration of substate which permits the enzyme to achieve 1/2 Vmax
Vmax definition
rate of reaction when the enzyme is saturated with the substrate is the maximum rate of reaction - Vmax
