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Biochemistry I Fall Term, 2004 November 12, 2004

Lecture 28: Carbohydrates 2

Lecture by Dr. Grotzinger

Assigned reading in Campbell: Chapter 13.4-13.5

Key Terms:

Cellulose (14) glucose

Starch (14) glucose

Glycogen (16) branch points

NAG (N-acetylglucosamine)

Lysozyme:

acid catalysis

transition state stabilization

oxonium ion

Links:

(I) Review Quiz on Lecture 28 concepts:

(I) Lysozyme Chemical Mechanism: Handout for Lecture 28.

(I) Trypsin and Lysozyme Mechanisms Compared: Handout for Lecture

(S) Lysozyme Binding & Catalysis: Structure is related to function on a Chime page.

Polysaccharides

Structural Polysaccharides

Cellulose: Structural polysaccharide of plants.Polymer of(14) glucose.

Similar in structure to -sheets in proteins: cellulose forms flat sheets with multiple

hydrogen bonds between strands.

Digested by symbiotic microorganisms.

Chitin: Structural polysaccharide in insect and shellfish exoskeletons.Contains N-acetylglucosamine (NAG).

Storage Polysaccharides

1. Starch [plants] (amylose and amylopectin):Amylose = Polymer of(14) glucose.

Similar in structure to -helix in proteins: forms a helix.

Amylopectin = amylose + (16) branches.

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2. Glycogen [animals]:More highly branched than starch.

Glucose units are released by the enzyme glycogen phosphorylase, producing

glucose-1-phosphate.

Glycoproteins & Glycosylated Proteins

Bacterial Cell Walls:

Polysaccharide chains of alternating NAG and NAM (N-acetylglucosamine + a lacticacid on O3).

NAG NAM 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 oligosaccharide 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-acetylglucosamine) 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 -galactosyl-(1,3)--N-

acetylgalactosyl attached to the OH of Ser or Thr.

Lysozyme Mechanism

14 kDa protein. Found in egg whites, tears, various other secretions.

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Disulfide bonded. Hydrolyzes the bond between NAG units in bacterial cell walls. May serve as a defence mechanism.

The rate of enzyme catalyzed hydrolysis is approximately 108

times that of the uncatalyzedreaction. 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. Gbinding for NAG3 = -24 kJ/mol4. Gbinding 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 O6atom of NAGD.

7. Glu 35 (pKa = 6.3!) protonates the oxygen in the glycosidic bond (between NAGD andNAGE (acid catalysis)

8. The glycosidic bond is broken, resulting in a carbocation on the C1 of NAGD.9. The carbocation is stabilized by formation of an oxonium ion. This requires the formation

of a planar, half-chair conformation of NAGD.

10.The carbocation is stabilized by the negative charge on Asp 53 (pKa=3.5, i.e. normal), buta 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 ionand the reprotonation of Glu 35.

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