Introduction to enzymes

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Introduction to enzymes
Ichiro Matsumura October 2, 2003
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Biochemists study the chemical reactions that
make life possible

• Most essential reactions are catalyzed by
  proteins called enzymes.

For example Prof. Pallas protein phosphatase
2A Prof. Edmondson monoamine oxidases Prof.
Corbett nuclear transport me enzyme evolution
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Enzymes catalyze nearly all chemical reactions in
a cell
Examples we've already encountered 1. DNA
polymerase 2. RNA polymerase 3. topoisomerase 4.
ribosomes These following are NOT enzymes 1.
sigma factors 2. single-strand DNA binding
protein 3. myoglobin and hemoglobin
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How do enzymes

• increase reaction rates?
• enable reactions under mild conditions?
• increase reaction specificity?
• enable regulation?

And how much do you guys need to know?
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Overview

• Review of free energy
• Catalysis
• Kinetics
• Inhibition
• Regulation
• Next time
• Basic metabolism and energetics

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Thermodynamics the study of Free Energy
Life is like a ball rolling down a hill. The
hill Free Energy The ball everything All
balls tend to roll down hill.
Free Energy
All things tend to fall into the state of lowest
free energy.
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Free Energy GChange in Free Energy DG
High G state - High energy - unstable.
Moving down, DG is negative, Favorable
Moving up, DG is positive, unfavorable - cant
do it without help.
Free Energy
Happiest of all - Lowest Free energy
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Pre-equilibrium
Mountain top
Mountain height
elevation
Valley depth
The valley of the balls
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Equilibrium
Mountain top
Mountain height
elevation
Valley depth
kF A kR B
The valley of the balls
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Chemical equilibria
transition-state
energy of activation
reactants
change in free energy
products
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Chemical free energy is a combination of
Enthalpy DH How much heat is absorbed Entropy
DS How much order is produced DG DH - T
DS Where T is the temperature. Thus, chemicals
release heat and/or become more disordered as
they lose free energy.
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The equilibrium constant
Keq B/A DGº -RT ln Keq
Keq describes the ratio of B to A at
equilibrium. Thus
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Enzymes accelerate reactions by decreasing DG
mountain top activation energy
Catalysts get the reaction to the equilibrium
faster, but dont affect Keq.
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Catalysis
Enzymes poise all the reactants in a arrangement
that allows the reaction to proceed more rapidly
than it would otherwise.
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Enzymes can stabilize all sorts of
transition-states

• Acid pKa conjugate base
• HCl -7 Cl-
• RCO2H 5 RCO2-
• H2CO3 6 HCO3-
• H2O 16 OH-
• CH4 62 CH3-

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Non-enzymatic catalysts also accelerate chemical
reactions
2 NO ? N2 O2 2 NO2 ?N2 2 O2 2 CO O2 ? 2 CO2
Platinum/rhodium surface of catalytic converter
accelerates these reactions
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Enzymes are more sophisticated catalysts

• Active Site
• Is complementary in shape and functionality with
  the substrate.
• Catalytic Efficiency
• Rate increased 103-108
• Specificity
• Reactions are extremely specific in both the
  substrates and the products.
• Regulation

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Enzymes increase reaction specificity
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Enzymes can be regulated
ATCase-CTP (inactive T state)
ATCase-ATP (active R state)
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Overview

• Review of free energy
• Catalysis
• Kinetics
• Inhibition
• Regulation

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How fast are enzyme catalyzed reactions?
In Liver, For when you eat
In other cells, For minute to minute energy
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Biochemists are interested in enzyme kinetics
because they enable

• the elucidation of catalytic mechanisms.
• the understanding of the roles of enzymes within
  metabolic processes.
• the optimization of enzyme-catalyzed processes
  (PCR, cloning and ELISA).

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Michaelis-Menten Equation
Simple reaction gives a hyperbolic curve for the
rate of the reaction vs. the substrate
concentration. Km reflects the affinity for the
substrate. Vmax reflects the rate of catalysis
and the concentration of enzyme.
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Active-site saturation
S gtgt KM v0 Vmax
SltltKM v0 Skcat/KM
S KM v0 1/2 Vmax
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The MM equation gets simple at the extremes.
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The Lineweaver-Burk plots estimate KM and Vmax
These parameters are on the X and Y intercepts.
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kcat and KM values vary a lot
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Competitive inhibition

• Inhibitor competes with substrate for binding to
  enzyme
• Example 1 most drugs
• Example 2 Product inhibition
• Weve made enough, so stop catalyzing!

E S ? ES ? E P
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Effect of Competitive Inhibition
Competitive inhibitors increase the apparent Km
of the substrate - That is they decrease the
effective affinity of the enzyme for the
substrate.
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Non-competitive inhibition

• Inhibitor inactivates the enzyme by binding to a
  site different than the substrate-binding site.
• Example
• lead poisoning

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Effects of Noncompetitive Inhibition
Noncompetitive inhibition inactivates a pool of
the enzyme - Thus reducing the effective
concentration of the enzyme.
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Allosteric regulation exhibits cooperativity
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Feedback inhibition of metabolic pathways
High concentrations of a downstream product of a
pathway signals an upstream enzyme to shut down.
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Regulation by covalent modification of an enzyme
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Overview

• Free energy which reactions are favorable?
• Catalysis how enzyme accelerate reactions
• Kinetics Michaelis-Menten equation
• Inhibition effect upon enzyme kinetics
• Regulation control of enzyme catalysis
• Next time
• Basic metabolism and energetics