Lecture 8 Flashcards

1
Q

Enzyme Kinetics
Why kinetics?
Some fundamental scientific reasons

A

• To establish the speed by which a reaction is catalyzed by an enzyme
– to understand how the enzymes in living organisms perform their functions in a timely fashion
• To see how catalysis is affected by factors like pH, ionic strength, temperature, etc. – to understand how different organisms are adapted to colder and warmer (etc.) environments
• To discover the substrate specificity of enzymes and ribozymes
• To figure out the mode of action of inhibitors – usually classified as
competitive, non-competitive and uncompetitive
• To understand accelerating or decelerating effects of non-substrate molecules on catalytic efficiency – particularly so-called allosteric effectors
• To probe in detail the catalytic mechanism of a reaction, not only by varying the substrates but also the biocatalyst itself, by e.g. protein engineering techniques and varying precisely specific side chains.

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2
Q

Enzyme Kinetics
Why kinetics?
Some practical reasons

A

• To establish the speed by which a reaction is catalyzed by an enzyme
or ribozyme – to see if a reaction is useful for industrial reactors to convert compounds into more valuable compounds
• To see how catalysis is affected by factors like pH, ionic strength, temperature, etc. – to obtain robust enzymes for bioreactors
• To discover, and modify, the substrate specificity of enzymes and ribozymes for use in bioreactors, and also as tools in biochemical and medical research
• To discover enzyme or ribozyme inhibitors in drug discovery processes, where sometimes thousands, or a few million, compounds are tested against a potential protein or RNA drug target in so-called “high-throughput screens”
• For lead optimization in drug development, i.e. optimize “leads” discovered in screens to become high affinity inhibitors with good “drug-like properties”.

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3
Q

Reaction rate is independent of ΔG

A

• ΔG describes the whether or not a reaction proceeds spontaneously and nothing about the rate of that reaction
• Some spontaneous reactions may not occur at physiological rates (e.g. peptide bond hydrolysis has a
half life of ~2500 years)
• Reaction rate depends on:
• In living systems, raising the temperature is not viable, thus, to increase reaction rates, must have ways to reduce ΔG0‡

Note: You don’t need to know this equation, but you need to know the dependence of k on ΔG0‡ and T

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4
Q

Enzymes increase reaction rates by lowering transition state energies

A
  • Enzymes reduce ΔG0‡ by stabilizing the transition state of the reaction
  • Reduction of transition state energy given by ΔΔG0‡ = ΔG0non - ΔG0cat

A catalyst lowers the activation energy in both directions

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5
Q

Enzyme Kinetic Experiment: S –> P

A
  1. Use constant amount of enzyme, [ET], where the T is for “total”.
  2. Measure amount of product P formed as a function of time with several initial concentrations of substrate S
  3. Calculate initial slopes from the graph of [P] vs. time to get initial velocities: vo = d[P]/dt
  4. Plot the initial velocities as function of [S]
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6
Q

Enzymes often form an Enzyme:Substrate complex

A

For many enzyme-catalyzed reactions:
• Acceleration is very fast (μsec to msec) while the time scale is in minutes.
• d[ES]/dt = 0 which means that a steady state condition applies to most of the reaction progress.

Total enzyme concentration [E]T is: [E]T = [E] + [ES]

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7
Q

The Michaelis-Menten Equation

A

The maximal velocity of a reaction, Vmax, occurs at high [S] when
the enzyme is saturated, that is, when [S]»KM

KM and Vmax are the quantities of interest
which capture the character of the catalysis of substrate S by enzyme E

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8
Q

Lineweaver-Burk plot

A

Then, this gives the Lineweaver-Burk plot with:
“ 1/[S] as the independent variable, and
“ 1/vo as the dependent variable.

This equation describes a straight line from which KM and Vmax are easy to obtain:
“ Slope: (KM/Vmax)
“ Intercept on the y-axis : 1/Vmax
“ Intercept on the 1/[S] axis: -1/KM

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9
Q

Competitive Inhibition

A

Inhibitor and Substrate compete for the Active Site

Ki is the quantity of
interest: it captures the inhibition power of inhibitor I for the particular reaction catalyzed by enzyme E.

Ki = [E][I] / [EI]
Hence: the SMALLER the Ki, the BETTER the inhibitor!

(E= enzyme, I= comp inhibitor, EI= enzyme-inhib complex)`

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10
Q

Uncompetitive Inhibition

A

Inhibitor binds to the enzyme-substrate complex, not to the free enzyme.

K’i is the quantity of interest: it captures the inhibition power of inhibitor I for the particular reaction catalyzed.

K’i = [ES][I] / [ESI]
Hence: the SMALLER the K’i, the BETTER the inhibitor!
(ES= micahaelis complex, I= uncomp inhib, ESI- enz-sub- inhib cmplex)

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11
Q

Uncompetitive Inhibition graph

A

Lineweaver-Burk Plot for Uncompetitive Inhibition: PARALLEL-LINES!!
With intersection points 1/v0= α’/Vmax on the vertical axis;
Since Vmax is known, α’ can be calculated;
And from α’, K’i can be calculated using the definition of α‘.

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12
Q

Mixed (noncompetitive) Inhibition

A

Inhibitor binds to both the enzyme-substrate complex and the free enzyme.

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13
Q

Protein Structure Prediction and Design

A

• Modern computers have allowed computational modeling of the free energy landscape of a protein

  • Represent the various enthalpic and entropic effects governing folding with parameterized equations
  • vdW interactions
  • electrostatic interactions
  • solvent entropy
  • etc.
  • Under Anfinsen’s hypothesis, the state of lowest free energy is the native state
  • Use various optimization techniques to identify the lowest-energy state of the protein
  • Design: identify a sequence that is predicted to fold into a particular state
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14
Q

De novo design of a novel coiled coil

A

• Natural coiled coils vary in their oligomerization state, but all known coiled coils are lefthanded
• Use computational design to create a right-handed coiled coils
1. Construct a backbone model for a right-handed coiled coil
2. Use sequence selection to optimize a sequence for this backbone

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