Enzymes II Dr. Kevin Ahern
E+S <=> ES <=> ES* <=> EP <=> E+P
Michaelis- Menten Kinetics E+S <=> ES <=> ES* <=> EP <=> E+P
Michaelis- Menten Kinetics Rate of Formation E+S <=> ES <=> ES* <=> EP <=> E+P
Michaelis- Menten Kinetics
Michaelis- Menten Kinetics Substrate
Michaelis- Menten Kinetics Substrate Product
Michaelis- Menten Kinetics Substrate Product Enzyme
Michaelis- Menten Kinetics Substrate Product Enzyme Enzyme- Substrate Complex
Michaelis- Menten Kinetics
Michaelis- Menten Kinetics Substrate high
Michaelis- Menten Kinetics Substrate high Enzyme high
Michaelis- Menten Kinetics Substrate high Enzyme high ES low Product low
Kinetic Considerations
Kinetic Considerations Pre-steady state [E] and [ES] varying widely
Kinetic Considerations Pre-steady state Steady state [E] and [ES] relatively constant [E] and [ES] varying widely
Pre- steady State Kinetics Can give info on reaction mechanism, rate of ES formation
Enzymes Kinetic Considerations
Enzymes Kinetic Considerations
Enzymes Kinetic Considerations Initial Velocity (V0) - Measured as [Product]/Time
Enzymes Kinetic Considerations Initial Velocity (V0) - Measured as [Product]/Time Substrate Concentration (Molarity)
Enzymes Kinetic Considerations Low [S], Low V0 (Enzymes Often Idle)
Enzymes Kinetic Considerations High [S], High V0 (Enzymes Almost Always Busy) Low [S], Low V0 (Enzymes Often Idle)
Kinetic Considerations
Michaelis- Menten Kinetics Parameters Km
Michaelis- Menten Kinetics Parameters Km = Substrate Concentration that Gives Vmax/2 NOT Vmax/2 Km
Michaelis- Menten Kinetics Parameters Km = Substrate Concentration that Gives Vmax/2 NOT Vmax/2 Km
Michaelis- Menten Kinetics Considerations v0 = Vmax[S] Km + [S] Enzymes That Don t Follow Michaelis-Menten Kinetics Include Those That Bind Substrate Cooperatively - Binding of One Substrate Affects Binding of Others
Control of Enzyme Activity Allosteric Control
Control of Enzyme Activity Allosteric Control
Control of Enzyme Activity Allosteric Control
Control of Enzyme Activity Allosteric Control Substrate Does Not Change Enzyme Binding of Substrate
Control of Enzyme Activity Allosteric Control Substrate Does Not Change Enzyme Binding of Substrate Substrate Does Change Enzyme Binding of Substrate
Michaelis- Menten Equation
Michaelis- Menten Equation Vmax occurs when an enzyme is saturated by substrate
Michaelis- Menten Equation Vmax occurs when an enzyme is saturated by substrate Vmax varies with amount of enzyme used
Michaelis- Menten Equation Vmax occurs when an enzyme is saturated by substrate Vmax varies with amount of enzyme used Km is a measure of an enzyme s affinity for its substrate
Michaelis- Menten Equation Vmax occurs when an enzyme is saturated by substrate Vmax varies with amount of enzyme used Km is a measure of an enzyme s affinity for its substrate Km value inversely related to affinity
Michaelis- Menten Equation Vmax occurs when an enzyme is saturated by substrate Vmax varies with amount of enzyme used Km is a measure of an enzyme s affinity for its substrate Km value inversely related to affinity High Km = Low Affinity Low Km = High Affinity
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes Since Vmax is a Velocity, and Velocity = [Product]/Time,
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes Since Vmax is a Velocity, and Velocity = [Product]/Time, Vmax [Enzyme Used] = [Product] [Enzyme Used] *Time
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes Since Vmax is a Velocity, and Velocity = [Product]/Time, Vmax [Enzyme Used] = [Product] [Enzyme Used] *Time The Two Concentrations Cancel Out.
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes Since Vmax is a Velocity, and Velocity = [Product]/Time, Vmax [Enzyme Used] = [Product] [Enzyme Used] *Time The Two Concentrations Cancel Out. The Result is a Number Per time (say 1000/second).
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes Since Vmax is a Velocity, and Velocity = [Product]/Time, Vmax [Enzyme Used] = [Product] [Enzyme Used] *Time The Two Concentrations Cancel Out. The Result is a Number Per time (say 1000/second). It is the Number of Product Molecules Made by Each Enzyme Molecule Per Time.
Michaelis- Menten Kinetics Vmax and Kcat Vmax is Proportional to the Amount of Enzyme Used in an Experiment - Not Useful for Comparing Enzymes Since Vmax is a Velocity, and Velocity = [Product]/Time, Vmax [Enzyme Used] = [Product] [Enzyme Used] *Time The Two Concentrations Cancel Out. The Result is a Number Per time (say 1000/second). It is the Number of Product Molecules Made by Each Enzyme Molecule Per Time. It is Also Known as the Turnover Number or Kcat and Does Not Vary with the Amount of Enzyme
Perfect Enzymes Maximum Kcat/KM Mutation leads to reduced Kcat/KM Diffusion of substrate limiting
Triose Phosphate Isomerase
Enzyme Co- factors
Michaelis- Menten Kinetics Lineweaver Burk Plot
Michaelis- Menten Kinetics Lineweaver Burk Plot Also Called Double Reciprocal Plot
Michaelis- Menten Kinetics Lineweaver Burk Plot Also Called Double Reciprocal Plot Uses Same Data as V vs. [S] Plot, but Inverts All Data for the Plotting
Michaelis- Menten Kinetics Lineweaver Burk Plot Also Called Double Reciprocal Plot Uses Same Data as V vs. [S] Plot, but Inverts All Data for the Plotting Linear for Enzymes Following Michaelis Menten Kinetics
Michaelis- Menten Kinetics Lineweaver Burk Plot Also Called Double Reciprocal Plot Uses Same Data as V vs. [S] Plot, but Inverts All Data for the Plotting Direct Reading of -1/Km and 1/Vmax
Michaelis- Menten Kinetics Lineweaver Burk Plot Also Called Double Reciprocal Plot Uses Same Data as V vs. [S] Plot, but Inverts All Data for the Plotting Direct Reading of -1/Km and 1/Vmax
Ribozymes
Metabolic Melody
Enzymes (To the tune of "Downtown") Copyright Kevin Ahern
Reactions alone Could starve your cells to the bone Thank God we all produce Enzymes Units arrange To make the chemicals change Because you always use Enzymes Enzymes (To the tune of "Downtown") Copyright Kevin Ahern Sometimes mechanisms run like they are at the races Witness the Kcat of the carbonic anhydrases How do they work? Inside of the active site It just grabs onto a substrate and squeezes it tight In an ENZYME! CAT-al-y-sis In an ENZYME! V versus S In an ENZYME! All of this working for you Energy peaks Are what an enzyme defeats In its catalysis Enzymes Transition state Is what an enzyme does great And you should all know this Enzymes Catalytic action won't run wild - don't get hysteric Cells can throttle pathways with an enzyme allosteric
Reactions alone Could starve your cells to the bone Thank God we all produce Enzymes Units arrange To make the chemicals change Because you always use Enzymes Sometimes mechanisms run like they are at the races Witness the Kcat of the carbonic anhydrases How do they work? Inside of the active site It just grabs onto a substrate and squeezes it tight In an ENZYME! CAT-al-y-sis In an ENZYME! V versus S In an ENZYME! All of this working for you Energy peaks Are what an enzyme defeats In its catalysis Enzymes Transition state Is what an enzyme does great And you should all know this Enzymes Catalytic action won't run wild - don't get hysteric Cells can throttle pathways with an enzyme allosteric Enzymes (To the tune of "Downtown") Copyright Kevin Ahern You know it's true So when an effector fits It will just rearrange all the sub-u-nits Inside an ENZYME! Flipping from R to T ENZYME! Slow catalytically ENZYME! No change in Delta G You should relax When seeking out the Vmax though There are many steps Enzymes Lineweaver Burk Can save a scientist work With just two intercepts Enzymes Plotting all the data from kinetic exploration Lets you match a line into a best fitting equation Here's what you do Both axes are inverted then You can determine Vmax and Establish Km for your ENZYMES! Sterically holding tight ENZYMES! Substrates positioned right ENZYMES! Inside the active site Enzymes (Enzymes, enzymes, enzymes)