Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2001
Lecture Notes:: 19 October	© R. Paselk 2001
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Enzyme Catalysis, cont.

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

As with general acid/base can account for a factor of about a hundred 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 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.

Enzyme Catalysis

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

Lysozyme: Have looked at model of Lysozyme - globular with cleft to accommodate substrate (overhead: V&V 14-10). Functions as an antibiotic, hydrolyzing polysaccharide strand in cell walls of bacteria. For Lysozyme the substrate is a carbohydrate polymer. (Figure 11-14, p300) [overhead 14-8, V&V].

Evidence:

• X-ray diffraction images show many H-bonding sites to substrate; Glu-35 and Asp-52 in active site near bond to be cleaved. (Figure 11-15, p301) [overhead 14-10, V&V]
• In binding studies find that binding energies increase as go from 1-4 residues, then decrease on 5 and increase again with 6.
• Lysozyme has a pH profile similar to papain with a pH optimum of 5, with Glu-35 unionized and Asp-52 ionized. Note pK's of both of these residues different than expected in free amino acids. Very typical in proteins due to local environments. Thus for Glu see pK shifted up because of local hydrophobic, non-polar environment (ions unstable in non-polar environment), whereas the Asp has an environment encouraging ionization.

So now let's look at the enzyme itself:

• Lysozyme uses a variety of catalytic mechanisms: General acid/base, TS strain distortion, & TS charge stabilization. Let's look at mechanism
  • Use binding energy to distort to half-chair conformation (C1 goes from sp³ to sp²-like geometry it will have in TS carbocation intermediate (Figure 11-16, p 303) [overhead 14-11, V&V].
  • Ionized (-) Asp-52 acts to stabilize (+) carbocation charge in TS. (Figure 11-19 p 305) [overheads: V&V 14-14, Horton Fig16.6]
  • Protonated Glu-35 acts as general acid (proton donor) to catalyze cleavage of glycosidic bond, then as general base (proton acceptor) to catalyze attack of water on carbocation intermediate. (Figure 11-19 p 305) [overheads: V&V 14-14, Horton Fig16.6]
  • In addition each of the catalytic groups is placed in close proximity and properly oriented, so we get proximity/orientation catalysis of catalysis.

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

• Asp-His-Ser catalytic triad in serine proteases (Figure 11-24, p 311). [overhead 66 V&V]
• Mechanism, as noted in Figure 11-26. [overhead 7.28, P]

Introduction to Vitamins and Cofactors

Terms:

• Cofactor: a co-catalyst required for enzyme activity.
  • coenzyme - a dissociable cofactor.
  • prosthetic group - non-dissociable cofactor.
  • metal ion - a metal cofactor.
• Vitamin: a required micro-nutrient (organism cannot synthesize adequate quantities for normal health - may vary during life-cycle).

Pathway Diagrams

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Last modified 19 October 2001