Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2008
Lecture Notes: 13 October	© R. Paselk 2008
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Enzyme Catalysis, cont.

Mechanisms of Chemical Catalysis, cont.

General acid/general base catalysis

(Recall that a 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 readily formed.

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

Look at specific base and specific acid catalysis first:

• Base catalysis of the mutarotation of glucose:

reverse to close as other conformer.

• Acid catalysis of the mutarotation of glucose:

reverse to close as other conformer.

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, respectively. Together they are better than either alone. But better yet is a concerted (both at once) catalyst with both functional groups present, so a three body collision isn't needed:

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

It turns out that a general acid or general base catalytic step can each account for about a 100-fold enhancement in explaining an enzyme's rate.

Covalent catalysis

Get formation of a covalent intermediate. Look at formation of methanol from methyl iodide and water. This reaction goes by an S_N2 mechanism:

Have a second order nucleophilic attack by hydroxide ion.

With Bromide catalyst get a new mechanism: formation of methyl bromide intermediate which is easier to form and to attack by hydroxide so reaction overall is faster.

Note the new lower activation energies. Only the higher one makes a difference in terms of rate. (Can tell that bromide intermediate is formed since, if we use chiral TDCHI as reactant, catalyzed product does not show inversion of chirality, while uncatalyzed does: must have double inversion.)

To summarize: The catalyst provides a new pathway (a new mechanism) in which all activation energies are lower so the rate determining step has a lower activation energy and thus the rate is faster.

As with general acid/base ctalysis, a covalent catalysis step can account for about a 100-fold enhancement in explaining enzyme rate.

Proximity/orientation

Example: [overhead 7.13, P].

Can get rate enhancements of up to a billion-fold in model systems. So Proximity/orientation can account for a factor of possibly a million to a billion-fold enhancement in explaining enzyme rate.

Stabilization of Transition State Conformation (Strain/distortion; Charge neutralization)

Strain/distortion example: Alkyl phosphate hydrolysis:

The base-catalyzed hydrolysis of A takes place more than one-hundred-million times faster than that of B, apparently due to the strain in the five membered ring in A.

Metal ion catalysis

Metal ions can act as electrophilic catalysts in covalent type catalytic mechanisms and also as counter ions in charge stabilization in TS catalytic mechanisms.

Example Enzymes

Now want to put it all together and look at example enzymes to try to explain their activities.

Chymotrypsin: (digestive enzyme: zymogens-precursor protein has peptide covering active site, activated by having it hydrolyzed off).

• Protein:
  • Fig 6-18a-d
  • Kinetics - presteady state vs. steady state: Fig 6-19
  • pH curve
    • a) overall bell-shape curve, need ionized His57 and protonated Ile16 for optimum activity.
    • b) rising leg shows titration of His57 as it ionizes, affecting catalysis
    • c) falling leg shows ionization of terminal Ile16 of B chain, affecting binding of the substrate due to enzyme conformation change as a salt-bridge is broken.
  • Asp-His-Ser catalytic triad in serine proteases - note general base catalysis of a general base catalysis of covalent attack for covalant catalysis of the hydrolysis of a peptide bond.
• Mechanism, as noted in text Figure 6-21, steps 1-7.

Pathway Diagrams

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Last modified 14 October 2008