Enzymes Flashcards
Lock-and-key hypothesis
Substrate-binding site exists in absence of substrate + fits chemically/geometrically w/enzyme’s substrate
Induced-fit hypothesis
Substrate binding triggers change in enzyme conformation to form final conformation of substrate-binding site - substrate engulfed by enzyme closing in on substrate
Active site
Substrate converted to product here
Cofactors
Assist enzymes when they act as catalysts - contain inorganic molecules or organic molecules (which makes them coenzymes)
Apoenzyme
Inactive form of enzyme observed before cofactor association
Holoenzyme
Active enzyme-cofactor complex
Prosthetic groups
Make up electron transport chain + tightly bound to proteins
5 ways enzymes can participate directly in the reaction mechanism
- Acid-base catalysis
- Covalent catalysis
- Metal ion catalysis
- Electrostatic catalysis
- Proximity + orientation effects
What explains the reaction rates achieved by enzymes along with the direct involvement of amino acid side chains in complex reaction mechanisms?
Enzymes preferentially bind to the activation complex X*
Steady-state assumption
Michaelis-Menten complex concentration is constant during rxn measurement time
Transient phase
Short initial time period required for initial diffusion of substrate to binding site
Turnover number (k2)
Number of rxns that each active site can catalyze /unit time once substrate has already been bound
Ligands
Molecules that attach to proteins via reversible non-covalent interactions - ligand bonding/covalent modifications of proteins causes shift in tertiary/quaternary structure of proteins
Enzyme regulators
Alter catalytic activity of enzymes
Enzyme activators
Increase catalytic activity upon binding the protein
Enzyme inhibitors
Decrease enzymatic activity upon binding
Feedback inhibition
Reduces flux through glycolysis pathway when end-product of pathway reaches elevated concentrations
Allosteric regulators
Don’t bind to active site of protein but change its activity by interacting w/remote site - act as activators/inhibitors
Homotropic
Term for allosteric effects if ligand at remote site is same as ligand at active site
Heterotropic
Term for allosteric effects if two ligands are different
Competitive inhibition
Reduces catalytic rates by competing w/substrate to enter + interact w/substrate-binding site
Uncompetitive inhibition
When inhibitors only have high affinity for enzyme-substrate complex
Mixed inhibition
When inhibitors can bind at allosteric sites both on free enzyme/on enzyme-substrate complex
Michaelis constant (Km)
Measurement of affinity of enzyme for substrate - higher Km leads to lower stability of ES complex
How do competitive inhibitors impact enzyme affinity for substrate + Vmax?
They decrease enzyme affinity for substrate + don’t alter Vmax
How do uncompetitive inhibitors affect the apparent Vmax and the apparent Km?
Decrease in apparent Vmax and Km - decrease in max rxn rate + increase in enzyme affinity for substrate
How do mixed inhibitors affect the apparent values of Km and Vmax?
Km apparent depends on the alpha/alpha’ ratio and Vmax apparent is decreased
Tense state vs relaxed state
Relaxed state has higher affinity to bind ligands/higher capacity to catalyze reactions than the tense state
Cooperative ligand binding
Change in affinity of 1 protein subunit for a given ligand depending on previous ligand-binding events occurring on other subunits - typically allosteric effect due to change in quaternary structure of protein
Positive cooperativity
Effect observed if binding a given ligand locks the protein in a relaxed state that increases binding affinity of protein for further ligands - more common than negative cooperativity
Negative cooperativity
Effect observed if affinity of protein for further ligands decreases after binding a 1st ligand
Hemoglobin
Protein that undergoes positive homotropic cooperative ligand binding - involved in oxygen transport from site where oxygen exchange occurs to other tissues + has 4 subunits including 2 alpha and 2 beta (subunits are analogous to myoglobin so 1 hemoglobin can bind 4 oxygen molecules in comparison to myoglobin that can only bind 1)
Myoglobin
Monomeric oxygen binding protein - its secondary structure has many alpha helices that fold together to form water soluble globular protein + contains 8 right-handed alpha helices that form compact structure
Heme group
- Compact structure in myoglobin contains this - hydrophobic pocket that tightly + non-covalently binds this
- Contains cyclic structure (porphyrin)
How does oxygen binding change the state of a protein?
Pressures a change from a tense (T) to a relaxed (R) state - T has lower oxygen binding affinity and R has higher so oxygen binding is cooperative (oxygen binding promotes further oxygen binding by pushing TR equilibrium towards R state - Perutz mechanism)
Fractional saturation
Ratio of occupied ligand-binding sites divided by total # of ligand-binding sites - in case of infinite cooperativity ignore the “ligand” term
What if Hill constant is greater than 1? Equal to 1? Less than 1?
- Greater: ligand binding is positively cooperative
- Equal: binding is non-cooperative
- Less: ligand binding to 1 subunit hinders binding to subsequent subunits
Intrinsic dissociation constant in the Adair model
Corresponds to dissociation constant of ith binding rxn between a ligand and an individual subunit
Macroscopic dissociation constant in the Adair model
Dissociation constant of ligand from any subunit on protein at ith binding step
How do changes in ki values in the Adair model indicate cooperativity?
Decrease of ki values as a function of i indicates positive cooperativity - increase of ki values as function of i indicates negative cooperativity + if k1 = k2= … kn then binding is non-cooperative
