Biochemistry Exam 2
uncompetitive (b/c it binds to the ES) and also noncompetitive (b/c can't be overcome by adding more substrate)
This model shows that you can observe at high [S]
uncompetitive
This model shows that you can usually see this type of inhibition with multisubstrates
enzyme
What cannot alter the equilibrium of the reaction but can increase the rate toward equilibrium?
gene
What do italicized enzymes refer to?
(1) reaction rates (2) substrate specificity & regulation (3) catalytic mechanism
What does enzyme kinetics provide information about?
-permanent -impairs enzyme activity through covalent interactions
What does irreversible mean?
at equilibrium the forward and reverse reactions are equivalent
What does it mean when a chemical process obeys the law of mass action?
They only work on one stereoisomer.
What does it mean when some enzymes are stereospecific?
-temporary -mimic transition state -effectively can be reversed by the substrate or by removing the inhibitor
What does reversible mean?
how fast a chemical process occurs
What does the rate of a reaction define?
Induced Fit Model
What does this describe? -catalysis -flexible -how binding a substrate leads to stabilizing of transition state for the reaction -The enzyme has a conformational change
The Lock & Key Model
What does this describe? -specificity -rigid -The enzyme-substrate complex has no conformational changes
catalyst
What enhances the rate of reaction but is not used up or permanently altered?
hexane kinase
What enzyme catalyzes the reaction: Glucose → Glucose-6-phosphate
Hsp90s
What finalizes the folding of a limited set of partially unfolded molecules?
metabolic reactions would not proceed at appreciable rates
What happens if enzymes didn't exist?
chaperones can promote protein degradation
What happens if refolding is not possible?
It denatures to an unfolded state, inactivating it. The disulfide cross-links are reduced to yield Cys residues.
What happens when urea and mercaptoethanol is added to the native state of ribonuclease A?
It goes back to the native state. It is catalytically active. The disulfide cross-links are correctly re-formed.
What happens when urea and mercaptoethanol is removed?
Urea disrupts H-bonds in protein and hydrophobic interactions. When combined with reducing agents, proteins unfold completely.
What happens when urea combines with a reducing agent?
chaperones
What helps proteins fold into their native conformation?
chaperones
What helps refold polypeptides?
Organic solvents
What interferes with hydrophobic interactions of proteins because they interact with nonpolar side chains and form H-bonds with water and polar side chains?
salting out
What is an effect based on the electrolyte-nonelectrolyte interaction, in which the non-electrolyte could be less soluble at high salt concentrations. It is used as a method of separating proteins. The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein.
hydrolysis of ATP coupled with metabolism
What is an example of a couple reaction?
bovine pancreatic ribonuclease
What is an example of reversible denaturation?
molten globule
What is an organized globular state of a polypeptide state of a polypeptide that resembles the molecule's native state (Tertiary interactions have not yet stabilized)?
A lowered ability of the blood to carry oxygen.
What is anemia?
The activation energy
What is energy required to convert the substrate to the transition state?
enzymes
What is important because organisms need to conserve energy and materials?
renaturation
What is it called when certain globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity if returned to conditions in which the native conformation is stable?
denaturation
What is it called when proteins partially or completely lose its biological activity?
Rate enhancement
What is it called when the catalyst accelerates the approach to equilibrium but not its position?
∆Gº
What is it called when the energy doesn't change regardless of a catalyst?
mechanical stress
What is it called when the stirring and grinding disrupt the forces that maintain protein structure?
Cysteine residues may react with heavy metals because of the high affinity between the sulfide and metal.
What is one way of heavy metal poisoning that can deform and inactivate the protein?
a consequence of protein aggregate formation as exposed hydrophobic surfaces associate. The aggregates are often highly disordered.
What is precipitation?
Disruption of protein structure. Denaturation is the loss of the three dimensional structure and it causes a loss of function.
What is protein denaturation?
environmental factors (physical and chemical agents)
What is protein structure sensitive to?
mechanical stress
What is referred to when the egg white is beaten to a denatured protein to form foam in meringue pie?
As for why denaturation tends to be irreversible as a rule, consider a familiar case. Think for a moment about frying an egg. The "white" before cooking isn't white; it's transparent and viscous. It's an aqueous solution of egg protein (ovalbumin) and a bit of salt. When you apply heat to the skillet, the ovalbumin is denatured, and the molecules stick to each other, forming a dense network. To the eye, you see that the transparent solution becomes white, opaque, and rubbery. The yolk, which also contains protein (in addition to fat and cholesterol), undergoes a similar transition from fluid to solid, for the same reason. Now, can you reverse this denaturation by letting the egg cool? A denatured protein represents a great many disrupted bonds and interactions. It is unlikely that all of those would reform in the same way to regenerate the original folded structure of the molecule.
What is reversible denaturation?
Salting out is when the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other.
What is salting out?
The activation energy
What is the difference between the energy levels of the ground state and the transition state?
Transition state
What is the highest energy state and the least stable?
The surface of the protein, in contact with water, will interact with lots of hydrophilic (polar) amino acid side chains, while the hydrophobic (nonpolar) amino acid side chains tend to be tucked away toward the protein's interior. When an organic solvent such as an alcohol is mixed with the aqueous solution, the amino acid side chains on the surface don't dissolve in the water-alcohol mixture as well. The protein molecule twists and flexes (through free bond rotations within) as the hydrophilic side chains shun the alcohol. At the same time, some interior hydrophobic side chains twist to the surface where they interact favorably with the organic solvent. The net effect is as though the protein molecule were trying to turn itself inside out in response to the change in the surrounding solvent. In the process H-bonds and ionic interactions will be broken. The result is a structurally altered, perhaps unraveled, protein molecule.
What is the hydrophobic effect in tertiary structure?
the pH at which a molecule carries no net electrical charge
What is the isoelectric point?
product
What is the product P?
kF = [A]^n
What is the rate expression of the forward reaction?
kR = [B]^m
What is the rate expression of the reverse reaction?
protein-protein interactions are formed
What is the result of salt concentration?
Rate enhancement
What is the same for forward and reverse reactions?
∆Gº
What is the same for the catalyzed and uncatalyzed reaction?
reactant
What is the substrate S?
favorable reaction
What is ∆Gº < 0?
unfavorable reaction
What is ∆Gº > 0?
protein folding
What isn't there a single pathway for?
denaturation
What may be reversible or irreversible?
heavy metals
What may disrupt salt bridges by forming ionic bonds with negatively charged side chains?
denaturation
What may or may not involve protein unfolding?
Ribosome-assisted chaperones
What prevents folding until the entire domain or polypeptide has emerged?
Chaperonins
What promotes fast and efficient refolding within an internal cellular compartment?
Hsp70s
What promotes folding of nascent (newly synthesized) polypeptides?
chaperones
What protects unfolded proteins from inappropriate hydrophobic protein-protein interactions that could cause misfolding or aggregration?
∆Gº≠
What represents the free energy of activation?
n & m
What represents the order of a reaction?
favorable reaction
What type of reaction is it if the free energy of the ground state of P is lower than that of S, so ∆Gº for the reaction is negative?
catalyst
What will not make an unfavorable reaction favorable?
temperature changes
When a transparent egg albumin is cooked it turns white. What is this an example of?
in its native state
When is ribonuclease A catalytically active?
competitive
This model shows that the substrate and inhibitor compete for the same site.
a compound that binds to an enzyme and interferes with activities. -It prevents the formation of the ES complex -It binds and competes with the substrate for the active site. -It noncovalently interacts
-The activity of an enzyme can be inhibited. What is an inhibitor?
noncompetitive
This model shows that they cannot be overcome by an increase in [S]
-ase
Enzymes end in what?
Purified ribonuclease A denatures completely in a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation. Denaturation of ribonuclease is accompanied by a complete loss of catalytic activity. When the urea and the reducing agent are removed, the randomly coiled, denatured ribonuclease spontaneously refolds into its correct tertiary structure, with full restoration of its catalytic activity.
Explain the denaturation of ribonuclease A.
-binding activators or inhibitors -covalent enzyme modification (posttranslation modifications (PTMs))
How can some enzymes be directly regulated?
-regulating synthesis of enzyme ex: can shut down enzyme function
How can some enzymes be indirectly regulated?
active site
How do enzymes increase the rate of reaction?
As temperature increases the rate of molecular vibrations increase and weak interactions (H-bonds) are disrupted.
How do temperature changes unfold proteins?
SDS's hydrocarbon tail dissolves any hydrophobic region of the protein, while the sulfate end breaks non-covalent ionic bonds. This causes the protein to lose its secondary and tertiary structure, and unfold.
How does SDS denature a protein?
Its amino acid composition is important for specific reactions
How is the active site more than just a binding site?
Rate of reaction increases significantly by a factor of 10^5 to 10^17.
How much does a rate of a reaction increase when enzymes are present?
Competitive
Inhibitor and substrate bind to the same site
noncompetitive
Inhibitor binds to a different location than the active site
Uncompetitive
Inhibitor binds to hidden ES complex site created -It appears only after the enzyme binds with substrate
noncompetitive
This model shows that little to no structural resemblance to substrate
competitive
This model shows that the affect of the inhibitor can be overcome by increasing the [S]
noncompetitive
This model shows that the binding site for the inhibitor is different from the binding site for the enzyme
uncompetitive
This model shows that the ineffective at low [S]
uncompetitive
This model shows that the inhibition cannot be overcome by adding more substrate
noncompetitive
This model shows that the inhibitor binds to a location on enzyme different from the active site
noncompetitive
This model shows that the inhibitor binds to free enzyme and the ES complex
competitive
This model shows that the inhibitor binds to free enzyme.
uncompetitive
This model shows that the inhibitor binds to the ES complex
competitive
This model shows that the inhibitor can be a transition state analog.
competitive
This model shows that the substrate and inhibitor are structurally similar.
organic solvents and detergents
What acts primarily by disrupting the hydrophobic interactions that make up the stable core of globular proteins?
active site E + S ←→ ES → E + P
What adopts the conformation of the transition state?
A very small subset of substrates
What are enzymes highly specific for?
promiscuous
What are enzymes referred to as when they are less specific for a class of substrates?
cofactors and coenzymes
What are nonprotein substances for catalysis?
enzymes
What are specific for substrates?
(1) oxidoreductases (2) transferases (3) hydrolases (4) lyases (5) isomerases (6) ligases
What are the 6 classes of enzymes?
(1) energy conservation (2) few to no side products in a reaction
What are the benefits to enzyme specificity?
-When [A] greatly excess [E] and reaction time is short -Assume P→A does not occur to an appreciable extent -Represents the rate of reaction at a known [A]
What are the characteristics of V0 (initial velocity)?
cofactors and coenzymes
What are the helpers of enzymes?
(1) Competitive (2) Uncompetitive (3) noncompetitive
What are the three types of reversible inhibitors?
(1) pure (2) mixed
What are the two types of noncompetitive inhibition?
the substrate or the type of reaction
What can an enzyme name tell you?
switch positions to accommodate catalysis
What can the residues in an enzyme do?
cofactors
What include Mg2+, Ca2+, Zn2+, and other ions?
coenzymes
What include more complex organic molecules such as NAD+, FAD, and AcCoA?
trypsin
What is an enzyme that is historical?
If n=2, then 2 molecules of A are needed to collide to form B.
What is an example of a second order reaction?
Enzyme Substrate [ES] Complex
What is called when the enzyme is bound to the substrate in the proper orientation (noncovalently) in the active site?
holoenzyme
What is it called when a protein does have a cofactor bound?
apoenzyme
What is it called when a protein does not have a cofactor bound?
shape and charge distribution
What will constrain certain motions and allow conformations of the substrate?
active site
What is the cleft/crevice where substrates bind in a specific orientation so that the reaction occurs (This promotes catalysis)
catalyst
What is the enzyme E?
( kF / kR )
What is the equilibrium constant Req?
kF [A]^n = kR [B]^m ( kF / kR ) = ( [B]^m / [A]^n )
What is the equilibrium reaction of the forward and reverse reaction?
ligases
What reaction does bond formation between two substrates?
isomerases
What reaction does intramolecular rearrangement?
lyases
What reaction generates or removes a double bond?
oxidoreductases
What reaction has oxidation/reduction reactions that both occur in the same reaction? -ex: NAD+ + 2H+ + 2e- → NADH + H+
hydrolases
What reaction hydrolyzes and cleaves a double bond with water?
transferases
What reaction transfers a group between two molecules?
reduction
What rxn gains electrons?
oxidation
What rxn loses electrons?
∆Gº
what represents Gibbs free energy or standard free-energy change?
∆∆Gº≠
∆Gºuncatalyzed≠ - ∆Gºcatalyzed≠
the protein molecules aggregate and precipitate. This process is usually reversible—used in protein purification.
What happens when the salt concentration is very high?
Chaperonins
What are GroEL in bacteria and Hsp60 in eukaryotes classified as?
Proteins that are not properly folded often have exposed hydrophobic surfaces that render them "sticky," leading to the formation of inactive aggregates.
What are aggregates?
enzymes
What are selective, efficient, and biological catalysts?
heat denaturation, a property that can be exploited for protein purification
What are some proteins more resistant to?
catalyst
What are substances that speeds up attainment of equilibrium?
(1) Ribosome-assisted chaperones (2) Hsp70s (3) Hsp90s (4) Chaperonins
What are the four groups of molecular chaperones?
Ribosome-assisted chaperones
What binds to the ribosome and emerging polypeptide?
reducing agents
What breaks disulfide bonds?
intermediates that are trapped in local energy wells
What can a polypeptide form depending on size?
heavy metals
What can bond to sulfhydryl groups, which changes protein structure and function?
enzymes
What can couple 2 reactions that normally occur separately?
Hsp70s
What can help refold misfolded and aggregated proteins and transfer to specific locations in the cell?
enzymes
What can respond quickly to small changes in a metabolic concentrated environment?
denaturation
What can we observe with the naked eye?
Hsp90s
What can work together with Hsp70 to identify proteins damaged by oxidative or heat stress and refold or target for degradation?
(1) Strong acids or bases (2) Organic solvents (3) Detergents (4) Reducing Agents (5) Salt concentration (6) Heavy metals (7) Temperature Changes (8) Mechanical Stress
What causes protein denaturation?
reducing agents
What converts disulfide bridges to sulfhydryl groups (beta-mercaptoethanol, dithiothreitol, TCEP)?
Hsp90s
What coordinates assembly of many protein complexes?
catalyst
What decreases the activation energy required for reaction ?
Alzheimer's, Huntington's, Mad Cow Disease
What diseases can misfolded proteins cause?
detergents
What disrupts the hydrophobic interactions of proteins causing them to unfold?
the protonation state of certain protein side chains
What do changes in pH alter?
a slower reaction
What does a higher activation energy correspond to?
Hsp90s
What does not bind nascent polypeptides?
catalyst
What does not change position of equilibrium?
denaturation
What does not involve breaking peptide bonds?
hydrophobic aggregation
What does precipitation of proteins occur by?
less soluble and may precipitate
What does the protein become as the pH of the solution reaches the isoelectric point?
H-bonding and salt bridges in proteins
What does the protonation state of certain side chains affect?
products dissociate
What happens when reactants bind to a catalyst?
Some water molecules that interact with the protein's ionizable groups are attracted to the salt ion. This causes a decrease in available water molecules to interact with protein.
What happens when salt concentration increases in an aqueous solution of protein?
Can cause lead poisoning.
Why are heavy metals potentially harmful?
they are strongly attracted to sulfur
Why do heavy metal salts disrupt disulfide bonds?
