Biochemistry I Fall Term, 2000

Lecture 29: Carbohydrates 2

Assigned reading in Campbell: Chapter 12.4-12.5

Take the Review Quiz on Lecture 29 concepts.

Lysozyme Binding & Catalysis: the structure is related to function on a Chime page.

Polysaccharides:

Structural Polysaccharides

1. Cellulose: Structural polysaccharide of plants.
   
   • b 1-4 glucose.
   • Similar in structure to b-sheets in proteins: forms flat sheets with multiple hydrogen bonds between strands.
   • Digested by symbiotic microorganisms.

2. Chitin: Structural polysaccharide in insect and shellfish exoskeletons.
   
   • contains N-acetylglucosamine.

Storage Polysaccharides

1. Starch [plants] (amylose and amylopectin).
   
   • amylose = a (1-4) glucose.
   • Similar in structure to a-helix in proteins: forms a helix.
   • amylopectin = amylose + a (1-6) branches.
2. Glycogen [animals]
   
   • more highly branched than starch.
   • glucose units released by the enzyme glycogen phosphorylase, producing glucose-1-phosphate.

Glycoproteins & Glycosylated Proteins

Bacterial Cell Walls:

• Polysaccharide chains of alternating NAG and NAM (N-acetylglucoseamine + a small peptide on O3)
• NAM peptide chains are crosslinked with pentaglycine bridges
• Synthesis of bacterial cell walls is inhibited by penicillin.

Eukaryotic Glycoproteins:

• Common form of protein modification (another is phosphorylation).
• Branched chain with a common core of saccharides.
• Attached oligo-saccharide chains are usually solvent exposed.
• They can be removed without large effects on protein structure.
• They are more disordered than the polypeptide part.
• Play an important role in directing intracellular location of protein ("protein trafficking").
• Define blood group (ABO) antigens.

• Two common linkages:

  1. N-linked: The first residue is usually NAG (N-acetyl glucosamine) attached to an Asn side chain in the sequence (Asn-X-Ser/Asn-X-Thr)

  2. O-linked:The first and second residues are usually b-galactosyl-(1, 3)-a-N-acetylgalactosyl attached to the OH of Ser or Thr .

Lysozyme Mechanism:

• 14 KDa protein.
• Found in egg whites, tears, various other secretions.
• Disulfide bonded.
• Hydrolyzes the bond between NAG subunits in bacterial cell walls.
• Serves as a defence mechanism.

The rate of enzyme catalyzed hydrolysis is approximately 10⁸ that of the uncatalyzed reaction. This enhancement is due to:

1. Binding of the substrate in a conformation that resembles the transition state.
2. Acid catalysis provided by a Glu residue.
3. Stabilization of the positive charge in the transition state by an ionized Asp residue.

Catalytic Mechanism (Residue numbers are for human lysozyme.):

1. A total of 6 NAG units fit in the active site cleft (subsites are designated "A-F").
2. A large number of interactions in the 'specificity pocket' define specificity for NAG.
3. DG_binding for NAG₃ = -24 kJ/mol
4. DG_binding for NAG₄ = +12.1 kJ/mol.
5. Therefore NAG bound in the active site is in a strained conformation - transition state stabilization.
6. This distortion is due to a hydrogen bond between the main chain of Val 110 and the O6 atom of NAG_D.
7. Glu 35 (pK_a = 6.3!) protonates the oxygen in the glycosidic bond (between NAG_D and NAG_E (acid catalysis)
8. The glycosidic bond is broken, resulting in a carbocation on the C1 of NAG_D.
9. The carbocation is stabilized by formation of an oxonium ion. This requires the formation of a planar, half-chair conformation of NAG_D.
10. The carbocation is stabilized by the negative charge on Asp 53 (pK_a=3.5, quite normal), but a covalent intermediate is not formed. This is an example of transition state stabilization in enzyme catalysis.
11. The reaction is completed by attack of a water molecule (hydroxide) on the oxonium ion and the reprotonation of Glu 35.

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