1020 CHAPTER 24 Catalysis
Tutorial:
Serine protease mechanismThe pocket in trypsin is narrow and has a serine and a negatively charged aspartate
carboxyl group at its bottom. The shape and charge of the binding pocket cause it to
bind long, positively charged amino acid side chains (Lys and Arg). This is why trypsin
hydrolyzes peptide bonds on the C-side of arginine and lysine residues. The pocket in
chymotrypsin is narrow and is lined with nonpolar amino acids, so chymotrypsin
cleaves on the C-side of amino acids with flat, nonpolar side chains (Phe, Tyr, Trp). In
elastase, two glycines on the sides of the pocket in trypsin and in chymotrypsin are re-
placed by relatively bulky valine and threonine residues. Consequently, only small
amino acids can fit into the pocket. Elastase, therefore, hydrolyzes peptide bonds on the
C-side of small amino acids (Gly and Ala).
The mechanism for bovine chymotrypsin-catalyzed hydrolysis of a peptide bond is
shown in Figure 24.7. The other serine proteases follow the same mechanism. The
reaction proceeds as follows:- As a result of binding the flat, nonpolar side chain in the pocket, the amide linkage
that is to be hydrolyzed is positioned very close to Ser 195. His 57 functions as a
general-base catalyst, increasing the nucleophilicity of serine, which attacks the
carbonyl group. This process is helped by Asp 102, which uses its negative charge
to stabilize the resulting positive charge on His 57 and to position the five-membered
ring so that its basic N atom is close to the OH of serine. The stabilization of a
charge by an opposite charge is called electrostatic catalysis. Formation of the
tetrahedral intermediate causes a slight change in the conformation of the protein
that allows the negatively charged oxygen to slip into a previously unoccupied area
of the active site known as the oxyanion hole. Once in the oxyanion hole, the neg-
atively charged oxygen can hydrogen bond with two peptide groups (Gly 193 and
Ser 195), which stabilizes the tetrahedral intermediate. - In the next step, the tetrahedral intermediate collapses, expelling the amino group.
This is a strongly basic group that cannot be expelled without the participation of
His 57, which acts as a general-acid catalyst. The product of the second step is an
acyl-enzyme intermediatebecause the serine group of the enzyme has been
acylated. (An acyl group has been put on it.) - The third step is just like the first step, except that water instead of serine is the nu-
cleophile. Water attacks the acyl group of the acyl-enzyme intermediate, with His 57
functioning as a general-base catalyst to increase water’s nucleophilicity and
Asp 102 stabilizing the positively charged histidine residue. - In the final step of the reaction, the tetrahedral intermediate collapses, expelling
serine. His 57 functions as a general-acid catalyst in this step, increasing serine’s
leaving tendency.
The mechanism for chymotrypsin-catalyzed hydrolysis shows the importance of
histidine as a catalytic group. Because the of the imidazole ring of histidine
is close to neutrality, histidine can act both as a general-acid catalyst and
as a general-base catalyst at physiological pH.
Much information about the relationship between the structure of a protein and its
function has been determined by site-specific mutagenesis, a technique that replaces one
amino acid of a protein with another. For example, when Asp 102 of chymotrypsin is
replaced with Asn 102, the enzyme’s ability to bind the substrate is unchanged, but its abil-
ity to catalyze the reaction decreases to less than 0.05% of the value for the native enzyme.
Clearly, Asp 102 must be involved in the catalytic process. We have seen that its role is to
position histidine and use its negative charge to stabilize histidine’s positive charge.CHCH 2COO−
side chain of an aspartate (Asp) residueCHCH 2CONH 2
side chain of an asparagine (Asn) residue1 pKa=6.0 2pKa3-D Molecule:
Chymotrypsin with bound
inhibitor