CHEMISTRY WESTERN Understanding Enzyme Kinetics and
Catalysis: Key Concepts exam with 100% accurate solutions,Western
Illinois University
Enzyme kinetic & catalysis
Michael Fu and Dr. Miles
1. How would the pKa of the side chain of aspartate be affected if the aspartate residue is brought into close proximity to glutamate residue?
pKa would increase.
2. How would the pKa of the side chain of histidine be affected if the histidine residue is brought into close proximity to glutamate residue?
pKa would increase
3. How would the pKa of the side chain of histidine be affected by moving the side chain from the surface of the protein into a hydrophobic pocket?
pKa would decrease
4. How would the pKa of the side chain of aspartate be affected if the aspartate residue is brought into close proximity to lysine residue? pKa would
decrease
An acid deprotonation rxn can be written in two ways:
1. HA + H2O <-> A- + H3O+ (i.e Asp side chain under
physiological pH) 2. HA+ + H2O <-> A + H3O+ (i.e. His’s side
chain under physiological pH)
A. If a proximal group that has a charge (+ or -):
,1. A positive charge group like Lys stabilizes the A- form so equilibrium favors the product HA is easier to deprotonate so it becomes a stronger
acid, thus a drop of pKa value. A negative charge group will destabilize A- so equilibrium favors reactant side.
2. A negative charge group like Glu stabilizes the HA+ form so equilibrium favors the reactant side HA+ is now harder to deprotonate so it becomes
a weaker acid, thus an increase of pKa value. A positive charge group will destabilize HA+ so equilibrium favors product side.
B. Coulomb’s interaction (dielectric constant)
Charge-charge interaction on the protein surface is usually weak due to insulation of bulk H2O, but this interaction will become stronger (thus more
favorable) when we move the charged group to the interior of protein. In other words, the charged form (A- in rxn 1 and HA+ in rxn 2) will be stabilized
and thus cause a shift of equilibrium (toward product in rxn 1, toward reactant in rxn 2) and then the pKa values (decrease of pKa for stronger acid;
increase of pKa for weaker acid) change.
Note: this is a gross simplification since people are doing research trying to interpret what could be the cause of pKa shift, which could depend on the
microenvironment of the protein interior structure (i.e. The pKa values of acidic and basic residues buried at the same internal location in a protein are
governed by di fferent factors – PMC).
What is catalytic power? How do you calculate it?
Substrate Speficity
Induced fit model
Stereospecificity
Be able to determine the pro R and pro S hydrogen of prochiral molecules.
What are Cofactors? Coenzymes? Prosthetic Groups?
What is a holoenzyme? Apoenzyme?
Be able to interpret Reaction coordinate diagrams. Know what the activation energy is. Free energy changes between substrates and products.
Identify intermediates and transition states, the rate determining step.
Know how enzymes increase the rates of the reaction.
Why is it bad to bind the substrate too tightly.
Why do enzymes bind the transition state tightly?
, Know what transition state analogs are?
Enzymes Catalytic power
Transition state binding/stabilization.
• Use the Michealis-Menten equation to calculate v, Vmax, Km or [S] and construct on graph paper and evaluate Lineweaver Burke plots . From the
graph determine Km, Vmax, kcat,and the specificity constant.
• Know the four types of reversible enzyme inhibition (Competitive, Noncompetitive, Mixed Noncompetitive, Uncompetitive) and can evaluate
reversible inhibitors by constructing Lineweaver Burke plots and evaluating their inhibition patterns. Know how the different types of inhibitors
affect the slope, yintercept, x-intercept.
• Given an inhibitor, the student should be able to take a data set, do the lineweaver burk plot, extrapolate the Km and Vmax for the uninhibited enzyme,
Identify which type of inhibitor by the inhibition pattern and determine the Ki.
• Can analyze a lineweaver burk plot for a multisubstrate enzyme and determine If it sequential or ping pong.
• Can recognize sigmodial velocity versus substrate concentration as cooperative binding. Recognize negative allosteric effectors and positive allosteric
effectors.
Enzymes Catalytic power
Transition state binding/stabilization.
Proximity effects
Acid-base catalysis
Know difference between specific acid-base catalysis ( Specific acid catalysis is by H+, Specific base catalysis is by OH-) and general acid-base
catalysis. pH profiles for general acid-base catalysis.
Know the mechanism of RNAase. The roles of the two histidines. The intermediate in the enzyme catalyzed reaction. Why RNAase doesn’t hydrolyze
DNA.
Covalent Catalysis
Nucleophilic catalysis
Metal Ion Catalysis
Catalysis: Key Concepts exam with 100% accurate solutions,Western
Illinois University
Enzyme kinetic & catalysis
Michael Fu and Dr. Miles
1. How would the pKa of the side chain of aspartate be affected if the aspartate residue is brought into close proximity to glutamate residue?
pKa would increase.
2. How would the pKa of the side chain of histidine be affected if the histidine residue is brought into close proximity to glutamate residue?
pKa would increase
3. How would the pKa of the side chain of histidine be affected by moving the side chain from the surface of the protein into a hydrophobic pocket?
pKa would decrease
4. How would the pKa of the side chain of aspartate be affected if the aspartate residue is brought into close proximity to lysine residue? pKa would
decrease
An acid deprotonation rxn can be written in two ways:
1. HA + H2O <-> A- + H3O+ (i.e Asp side chain under
physiological pH) 2. HA+ + H2O <-> A + H3O+ (i.e. His’s side
chain under physiological pH)
A. If a proximal group that has a charge (+ or -):
,1. A positive charge group like Lys stabilizes the A- form so equilibrium favors the product HA is easier to deprotonate so it becomes a stronger
acid, thus a drop of pKa value. A negative charge group will destabilize A- so equilibrium favors reactant side.
2. A negative charge group like Glu stabilizes the HA+ form so equilibrium favors the reactant side HA+ is now harder to deprotonate so it becomes
a weaker acid, thus an increase of pKa value. A positive charge group will destabilize HA+ so equilibrium favors product side.
B. Coulomb’s interaction (dielectric constant)
Charge-charge interaction on the protein surface is usually weak due to insulation of bulk H2O, but this interaction will become stronger (thus more
favorable) when we move the charged group to the interior of protein. In other words, the charged form (A- in rxn 1 and HA+ in rxn 2) will be stabilized
and thus cause a shift of equilibrium (toward product in rxn 1, toward reactant in rxn 2) and then the pKa values (decrease of pKa for stronger acid;
increase of pKa for weaker acid) change.
Note: this is a gross simplification since people are doing research trying to interpret what could be the cause of pKa shift, which could depend on the
microenvironment of the protein interior structure (i.e. The pKa values of acidic and basic residues buried at the same internal location in a protein are
governed by di fferent factors – PMC).
What is catalytic power? How do you calculate it?
Substrate Speficity
Induced fit model
Stereospecificity
Be able to determine the pro R and pro S hydrogen of prochiral molecules.
What are Cofactors? Coenzymes? Prosthetic Groups?
What is a holoenzyme? Apoenzyme?
Be able to interpret Reaction coordinate diagrams. Know what the activation energy is. Free energy changes between substrates and products.
Identify intermediates and transition states, the rate determining step.
Know how enzymes increase the rates of the reaction.
Why is it bad to bind the substrate too tightly.
Why do enzymes bind the transition state tightly?
, Know what transition state analogs are?
Enzymes Catalytic power
Transition state binding/stabilization.
• Use the Michealis-Menten equation to calculate v, Vmax, Km or [S] and construct on graph paper and evaluate Lineweaver Burke plots . From the
graph determine Km, Vmax, kcat,and the specificity constant.
• Know the four types of reversible enzyme inhibition (Competitive, Noncompetitive, Mixed Noncompetitive, Uncompetitive) and can evaluate
reversible inhibitors by constructing Lineweaver Burke plots and evaluating their inhibition patterns. Know how the different types of inhibitors
affect the slope, yintercept, x-intercept.
• Given an inhibitor, the student should be able to take a data set, do the lineweaver burk plot, extrapolate the Km and Vmax for the uninhibited enzyme,
Identify which type of inhibitor by the inhibition pattern and determine the Ki.
• Can analyze a lineweaver burk plot for a multisubstrate enzyme and determine If it sequential or ping pong.
• Can recognize sigmodial velocity versus substrate concentration as cooperative binding. Recognize negative allosteric effectors and positive allosteric
effectors.
Enzymes Catalytic power
Transition state binding/stabilization.
Proximity effects
Acid-base catalysis
Know difference between specific acid-base catalysis ( Specific acid catalysis is by H+, Specific base catalysis is by OH-) and general acid-base
catalysis. pH profiles for general acid-base catalysis.
Know the mechanism of RNAase. The roles of the two histidines. The intermediate in the enzyme catalyzed reaction. Why RNAase doesn’t hydrolyze
DNA.
Covalent Catalysis
Nucleophilic catalysis
Metal Ion Catalysis
