Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2001
Lecture Notes:: 17 October	© R. Paselk 2001
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TEMPERATURE AND pH EFFECTS ON ENZYMES (AND PROTEINS)

Temperature profile reflects two underlying phenomena:

• The effects of temperature on rate due to the fact that as temp. increases more molecules will have the energy required to achieve activation energy, E_a. (Review: Arrhenius equation: , and the Maxwell-Boltzman Distribution.)
• The effects of temperature on protein stability: at high temperatures the protein denatures.

Together these effects lead to the plot below where the rising leg is due to activation energy effects (increasing rate) and the falling leg is due to protein denaturation.

pH EFFECTS ON ENZYME RATE

Papain: inflection at pH 4.2 for cys-25 and at pH 8.2 for his-159.

Note that the two legs represent two pH titration curves (rotate the left leg 90 deg. then flip; rotate the right leg 90 deg. counter clockwise and you can see them), with pK's equal to 4.2 and 8.2 respectively. This is a typical example for an enzyme with titratable groups in the active site. Can also have non-symmetrical curves with only one group. And of course can have curves due to denaturation by titration of charged surface and interior side chains.

Enzyme Catalysis

We will look at catalysis in two types of systems:

• Model systems in organic chemistry to elucidate probable mechanisms of chemical catalysis.
• Example enzymes to demonstrate these mechanisms in enzyme catalysis.

Mechanisms of Chemical Catalysis

Look at some examples of catalysis in model systems (organic chemistry) and how they might operate in enzymes.

Types of Catalysis:

• Acid/Base
  • Specific (H⁺ & OH^- in water)
  • General (Bronsted acid definition: proton donor/acceptor pairs; buffers.)
• Covalent
  • nucleophilic
  • electrophilic
• Proximity/orientation
• Stabilization of Transition State Conformation (Strain/distortion; Charge neutralization)
• Metal/Metal ion

General acid/general base catalysis: (General acid is a proton donor, Bronsted acid definition, such as a buffer, other than the solvent. Specific acid or base is the solvent acid or base species: H⁺ & OH^- for water). What we want is a proton donor (or acceptor) which is present at reasonable concentration at pH 7! Look at specific base and acid catalysis first:

So how does catalysis work? Recall that the slow step of a reaction is reaching the transition state. Thus if we can find a way to stabilize the transition state (lower E_a) then the reaction rate will be enhanced. Generally we will be looking tree ways to increase rates

1. stabilize transition states
2. increase the concentrations of intermediates
3. use a different reaction pathway.

General acid/general base catalysis: (General acid is a proton donor, Bronsted acid definition, such as a buffer, other than the solvent. Specific acid or base is the solvent acid or base species: H⁺ & OH^- for water). What we want is a proton donor (or acceptor) which is present at reasonable concentration at pH 7!

Consider the mutarotation of glucose:

This reaction will be slow because

1. the anomeric -OH is a very weak acid (it doesn't dissociate easily), and
2. the resulting alkoxide ion is very unstable and thus not readilty formed.

So the question is, How do we catalyze this process?

 

Look at specific base and acid catalysis first:

• Base catalysis of the mutarotation of glucose:

• Acid catalysis of the mutarotation of glucose:

Note that both are useful, but only one or the other can be used with specific acid or base catalysts. Would be nice to use both simultaneously.

In organic solvents can use phenol,, and pyridine,, as general acid and base catalysts. Together they are better than either alone. But better yet is a concerted catalyst with both functional groups present, so don't need a three body collision:

This is about 7,000 x's faster than separate species at 0.001M. (Studies in organic media complicated by fact that can get charge stabilization catalysis, so not necessarily looking at pure effects.)

A general acid/base catalytic step can account for a factor of about a hundred fold enhancement in explaining enzyme rate.

Pathway Diagrams

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Last modified 17 October 2001