Enzymes II Dr. Kevin Ahern

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

E+S <=> ES <=> ES* <=> EP <=> E+P Michaelis-Menten Kinetics Rate of Formation E+S <=> ES <=> ES* <=> EP <=> E+P

Product Substrate Enzyme Enzyme- Substrate Complex Michaelis-Menten Kinetics Substrate Product Enzyme Enzyme- Substrate Complex

Substrate high Enzyme high ES low Product low Michaelis-Menten Kinetics Substrate high Enzyme high ES low Product low

Kinetic Considerations 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

Enzymes High [S], High V0 (Enzymes Almost Always Busy) Kinetic Considerations Initial Velocity (V0) - Measured as [Product]/Time Low [S], Low V0 (Enzymes Often Idle) Substrate Concentration (Molarity)

Kinetic Considerations

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 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 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 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

Mutation leads to reduced Kcat/KM Diffusion of substrate limiting 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 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

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   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 V versus S All of this working for you Energy peaks Are what an enzyme defeats In its catalysis Transition state Is what an enzyme does great And you should all know this 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 Slow catalytically 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 Substrates positioned right Inside the active site Enzymes (Enzymes, enzymes, enzymes) Metabolic Melody