Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

42 Chapter 3
O
O
Glu 143
O −
R
H
H
O O
Zn 2+
H
N
O
Ph
Glu 143
O
R
HO
H
Zn 2+
O−
H
N
O
Ph
O
Glu 143
O −
R
H 2 N
OH
O
O
Ph
Zn 2+
Figure 3.16 Mechanism for thermolysin. R, peptide chain.
Second, since enzyme active sites are often largely excluded from water
molecules, an enzyme active site nucleophile is likely to be ‘desolvated’. Thus,
a charged nucleophile in aqueous solution would be surrounded by several
layers of water molecules, which greatly reduce the polarity and eVectiveness
of the nucleophile. However, a desolvated nucleophile at a water-excluded
active site will be a much more potent nucleophile than its counterpart in
solution. This eVect can be illustrated in organic reactions carried out in dipolar
aprotic solvents such as dimethylsulphoxide (DMSO) or dimethylformamide
(DMF), in which nucleophiles are not hydrogen bonded as they would be in
aqueous solution. A consequence of this desolvation eVect is that nucleophilic
displacement reactions occur much more readily in these solvents.
Enzymes have a range of potential nucleophiles available to them, which are
shown in Table 3.1.
Probably the best nucleophile available to enzymes is the thiol side chain of
cysteine, which we shall see operating in proteases and acyl transfer enzymes.
The e-amino group of lysine is used in a number of cases to form imine linkages
with ketone groups in substrates, as in the example of acetoacetate decarboxylase,
shown in Figure 3.17.
This enzyme catalyses the decarboxylation of acetoacetate to acetone. Two
lines of evidence were used to show that an imine linkage is formed between the
ketone of acetoacetate and the e-amino group of an active site lysine. First,