Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Enzymatic Hydrolysis and Group Transfer Reactions 89
ment of functional groups is especially adept for this type of catalysis.
The speciWcity of trypsin is for cleavage after basic amino acids such as lysine
and arginine. This speciWcity is provided by a similar speciWcity pocket to that of
chymotrypsin in which there is the carboxylate side chain of Asp-189 at the bottom
of the pocket which forms a favourable electrostatic interaction with the basic side
chains of lysine and arginine-containing substrates (see Figure 5.3).
There are several other classes of enzyme which also contain serine catalytic
triads. The ab-hydrolase family of esterase and lipase enzymes, discussed in
Section 5.3, also contain active site serine groups, and use the same type of
mechanism as chymotrypsin (which itself is capable of hydrolysing esters as well
as amides).
The cysteine proteases
This family of proteins is characterised by an active site cysteine residue whose
thiol side chain is also involved in covalent catalysis. The cysteine proteases are
less commonly used for digestive purposes and are more often found in intracellular
proteases used for post-translational processing of cellular proteins.
The active site thiol is prone to oxidation, which means that these enzymes must
be puriWed and handled in the presence of mild reducing agents. The active site
cysteine is easily modiWed by cysteine-directed reagents such as p-chloromercuribenzoate,
an organomercury compound which functions by forming a
strong mercury–sulphur bond.
The best characterised member of this family is papain, a 212-amino acid
endoprotease found in papaya plants. The preferred cleavage site is following
basic amino acids such as arginine and lysine. The active site of papain contains
the nucleophilic Cys-25 and an active site base, His-159, as shown in Figure 5.8.
There is good evidence that Cys-25 acts as a nucleophile to attack the amide
bond, generating a covalent thioester intermediate. There is evidence from
X-ray crystallography and modiWcation studies that Cys-25 is deprotonated
by His-159 as it attacks the amide substrate. As in the case of the serine
proteases, a high-energy oxyanion intermediate is formed which is speciWcally
stabilised by hydrogen bonding to the backbone amide N2H bonds of Cys-25
and Gln-17. Breakdown of the thioester intermediate by base-catalysed
attack of water leads to formation of the carboxylic acid product, as shown
in Figure 5.9.
Analysis of the active site histidine residue by 1 H nuclear magnetic resonance
(NMR) spectroscopy has revealed that in the active form of the enzyme
the imidazole ring is in fact protonated, suggesting that in this case the resting
state of the enzyme contains an imidazolium–thiolate ion pair. This is possible
in the case of the cysteine proteases since the pK a of the thiol side chain of
cysteine is only 8–9, and stabilisation of the ion pair by active site electrostatic
interactions seems likely.