Organic Chemistry

PROBLEM 17

Arginine and lysine side chains fit into trypsin’s binding pocket. One of these side chains

forms a direct hydrogen bond with serine and an indirect hydrogen bond (mediated

through a water molecule) with aspartate. The other side chain forms direct hydrogen

bonds with both serine and aspartate. Which is which?

PROBLEM 18

Serine proteases do not catalyze hydrolysis if the amino acid at the hydrolysis site is a

D-amino acid. Trypsin, for example, cleaves on the C-side of L-Arg and L-Lys, but not on

the C-side of D-Arg and D-Lys. Explain.

Mechanism for Lysozyme

Lysozyme is an enzyme that destroys bacterial cell walls. These cell walls are composed

of alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) units.

Lysozyme destroys the cell wall by catalyzing the hydrolysis of the NAM–NAG bond.

The active site of hen egg-white lysozyme binds six sugar residues of the substrate.

The many amino acid residues involved in binding the substrate in the correct position

in the active site are shown in Figure 24.8. The six sugar residues are labeled A, B, C,

D, E, and F. The carboxylic acid substituent of NAM cannot fit into the binding site for

C or E. This means that NAM units must be in the sites for B, D, and F. Hydrolysis oc-

curs between D and E.

Lysozyme has two catalytic groups at the active site: Glu 35 and Asp 52 (Figure 24.9).

Once it was determined that the enzyme-catalyzed reaction takes place with retention of

configuration at the anomeric carbon, it could be concluded that it cannot be a one-step

reaction; the reaction must involve at least two steps and, therefore, must form an

intermediate. Although lysozyme was the first enzyme to have its mechanism studied—

and the mechanism has been studied extensively for almost 40 years—only recently

have data been obtained that support the mechanism shown in Figure 24.9:

• In the first step of the reaction, Asp 52 acts as a nucleophilic catalyst and attacks
  C-1 of the NAM residue, displacing the leaving group. Glu 35 acts as a general-
  acid catalyst, protonating the leaving group and thereby making it a weaker base

SN 2

+

R = CH 3 CH H 2 O

COO−

CO

CH 3

CH 2 OH

CH 2 OH

CH 2 OH

CH 2 OH CH 2 OH

CH 2 OH

O O

HO

NH

CO

CH 3

O O

RO

NH

CO

CH 3

O

O

O

HO

NH

NAG

NAG

NAM

CO

CH 3

O O

HO

NH

CO

CH 3

O O

RO

NH

CO

CH 3

OH

HO

O

O

HO

NH

NAG

NAM NAG

lysozyme-catalyzed

hydrolysis occurs here

1022 CHAPTER 24 Catalysis