Enzyme Kinematics - Notes PDF

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Notes...

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Michaelis-Menten: ● ●

Plotting reaction speed (Y axis) vs Substrate concentration (X axis) Two conditions: ○ Enzyme concentration is held constant ○ Substrate concentration is increased (what you are changing to see effect)

Reaction Velocity (V0)

A

B

Substrate Concentration [s] C A: Is the Vmax. B: Is ½ Vmax C: Is Km ●

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At really high [S] the enzyme will be saturated ○ Even if you increase the concentration of [S] from this point, there will still be a Vmax Vmax: Maximum enzyme velocity ○ Vmax = [E]Kcat ○ The only way to increase the max is by increasing the enzyme concentration Kcat: Enzyme turnover rate ○ The number of substrate molecule each enzyme site can convert to product in a unit of time (single enzyme efficiency) Km: the substrate concentration at which the reaction rate is equal to ½ Vmax ○ Km is known as the Michaelis constant and can be used to compare enzymes because it is a measure of affinity of the enzyme for its substrate

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■ Lower Km = higher affinity Km is an intrinsic property of the enzyme-substrate system and cannot be altered by changing the concentration of substrate or enzyme

Michaelis Menten Equation and other important equations: ○ V=Vmax [S]/Km + [S] ○ Vmax = [E]* Kcat (same as above) ○ Kcat/Km = catalytic efficiency

Lineweaver Burke: ●

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Same data as MM plot, but graphed on a different scale: taking the reciprocal of each axis ○ X axis changes from [S] to 1/[S] and Y axis changes from V0 to 1/V0. This makes the graph linear This shows the Km and Vmax ○ The Y intercept (where the graph line crosses the Y axis, when x = 0) is 1/Vmax and the X intercept (where the graph line crosses the X axis, when Y =0) is -1/Km

A B

A: 1/Vmax B: -1/Km The Line-weaver Burke plot is very useful for reversible inhibition (very important to know for the mcat)

Reversible Inhibition types: ●

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Competitive inhibition ○ Involves the occupancy of the active site ○ Substrates cannot access the enzymatic binding sites ○ Can be overcome by adding more substrate so the substrate to inhibitor ratio is higher ○ Does not alter the value of Vmax ■ Can overcome the inhibitor but increasing more substrate ○ Increases the value of Km ■ Inhibitor is potentially blocking the binding site so the substrate will not be able to bind as often, thus making the affinity worse (so increases Km) Uncompetitive Inhibition ○ Inhibitor binds only to the enzyme-substrate complex at allosteric site ○ Vmax decreases ■ The enzyme will not be able to work as fast because the ES complex has been bound by the inhibitor ○ Km decreases ■ As inhibitors bind to the ES complex, the amount of uninhibited ES complex decreases. This causes an increase in affinity between the substrate and the enzyme as they need to increase the amount of enzyme-substrate complex there is (Le Chatelier’s principle) so the affinity will increase Mixed Inhibition ○ Binding of allosteric site ○ Can bind to both enzyme and enzyme substrate complex, but not equally well (equally well would be Non competitive Inhibition) ○ Vmax decreases ■ Inhibitor binds to decrease turnover rate ○ Km can either increase or decrease depending on whether it binds to enzyme substrate complex of enzyme ■ If ES more → decrease Km ■ If Enzyme more → increase Km Non Competitive Inhibition ○ Special case of Mixed Inhibition ○ Binding of allosteric site ○ Bind equally well to the enzyme and enzyme substrate complex ○ Decreases Vmax ■ Inhibitor binds to ES complex and will decrease the turnover ○ Does not affect Km ■ Because it binds equally well to the enzyme and ES complex ● There is a decrease in Km for if it binds to the ES complex and an increase in Km for if it binds to the enzyme so no change

Vmax

Km

Binding Site

Competitive

Same

Increases

Active

Uncompetitive

Decrease

Decreases

Allosteric only on ES

Mixed

Decrease

Increase or Decrease

Allosteric on either E or ES, one more favored

Noncompetitive

Decrease

Same

Allosteric on E or ES (50/50 preference)

Lineweaver Burke with inhibitors Competitive

Red line is inhibited

Uncompetitive

Red line is inhibited

Non Competitive

Red line is inhibited

Cooperativity:

○ Cooperativity ■ Some proteins can bind more than one substrate ■ Cooperativity-substrate binding changes substrate affinity ■ Positive cooperative binding- substrate binding increases affinity for subsequent substrate ■ Negative cooperative binding - substrate binding decreases affinity for subsequent substrate ■ Non cooperative binding- substrate binding does not affect affinity for subsequent substrate ■ Ex: Hemoglobin, Tense state = low affinity, Relaxed state = high affinity ■ Sigmoidal kinetics ■ Hills coefficient ● Hill coefficient = 1 = no cooperative binding ● Hills coefficient 1 = positively cooperative Cooperativity Graph: Sigmoidal

Reaction Velocity (V0)

Substrate Concentration [s]...