enzymes ii - oregon state...

Enzymes IIDr. Kevin Ahern

E+S <=> ES <=> ES* <=> EP <=> E+P

Michaelis-‐Menten Kinetics

E+S <=> ES <=> ES* <=> EP <=> E+P


E+S <=> ES <=> ES* <=> EP <=> E+P

Rate of Formation

Substrate


Substrate Product


Substrate Product

Enzyme


Substrate Product

Enzyme

Enzyme- Substrate Complex



Substrate high


Substrate high

Enzyme high


Substrate high

Enzyme high

ES low Product low

Kinetic Considerations


Pre-steady state

[E] and [ES] varying widely


Pre-steady state

[E] and [ES] varying widely

Steady state

[E] and [ES] relatively constant

Pre-‐steady State Kinetics

Can give info on reaction mechanism, rate of ES formation

Enzymes• Kinetic Considerations


Initial Velocity (V0) - Measured as [Product]/Time


Initial Velocity (V0) - Measured as [Product]/Time

Substrate Concentration (Molarity)


Low [S], Low V0 (Enzymes Often Idle)


Low [S], Low V0 (Enzymes Often Idle)

High [S], High V0 (Enzymes Almost Always Busy)

Michaelis-‐Menten Kinetics• Parameters

Km

Michaelis-‐Menten Kinetics• Parameters

Km

Km = Substrate Concentration that Gives Vmax/2 NOT Vmax/2

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


Substrate Does Not Change Enzyme Binding of Substrate


Substrate Does Not Change Enzyme Binding of Substrate Substrate Does Change Enzyme

Binding of Substrate

Michaelis-‐Menten Equation


Vmax occurs when an enzyme is saturated by substrate


Vmax occurs when an enzyme is saturated by substrateVmax varies with amount of enzyme used


Vmax occurs when an enzyme is saturated by substrateVmax varies with amount of enzyme usedKm is a measure of an enzyme’s affinity for its substrate


Vmax occurs when an enzyme is saturated by substrateVmax varies with amount of enzyme usedKm is a measure of an enzyme’s affinity for its substrateKm value inversely related to affinity


Vmax occurs when an enzyme is saturated by substrateVmax varies with amount of enzyme usedKm is a measure of an enzyme’s affinity for its substrateKm 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



Since Vmax is a Velocity, and Velocity = [Product]/Time,


Vmax

[Enzyme Used]= [Product]

[Enzyme Used] *Time




Vmax


[Enzyme Used] *Time


The Two Concentrations Cancel Out.



Vmax


[Enzyme Used] *Time


The Two Concentrations Cancel Out. The Result is a Number Per time (say 1000/second).



Vmax


[Enzyme Used] *Time



It is the Number of Product Molecules Made by Each Enzyme Molecule Per Time.



Vmax


[Enzyme Used] *Time



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


Also Called Double Reciprocal 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



Direct Reading of -1/Km and 1/Vmax

Ribozymes

Metabolic Melody

Enzymes (To the tune of "Downtown")

Copyright Kevin Ahern

http://www.davincipress.com/professional.html


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

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


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)


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

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 ii - oregon state...

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