Chemistry Reference
In-Depth Information
3.4
Active Site Considerations
Enzymes have evolved to accept substrates with particular molecular geometries
and functional groups. Part of this specificity can be attributed to the local environ-
ments of the secondary binding pockets located within or proximal to the active
site. There are many evolutionary conserved residues shared between trypsin and
α-chymotrypsin, but it is their differences that permit the binding of different sub-
strates. The
S1
binding pockets of the serine proteases are formed by residues 189-
195, 214-220, and 225-228 [
28
] of which four of these residues are not conserved
between trypsin and α-chymotrypsin; residues 189, 192, 217 and 219. For exam-
ple Ser
190
is differently oriented within each enzyme; pointing into the
S1
binding
pocket of trypsin but in α-chymotrypsin it points away from the
S1
binding pocket
where it hydrogen bonds to Thr
138
[
28
]. The bottom of the
S1
site in trypsin and
α-chymotrypsin show an additional dissimilarity; trypsin contains an Asp
189
which
is anionic at neutral pH and stabilizes the positive charge of the side chains of lysine
and arginine residues [
29
]. The
S1
binding pocket of α-chymotrypsin, on the other
hand, does not contain this anionic group allowing it to accept hydrophobic amino
acid side chains [
30
].
Pepsin, on the other hand, is a carboxypeptidase and contains two binding pockets
adapted for aromatic rings that are located proximally to the catalytic carboxylic
acid residues, Asp
215
and Asp
32
. These residues serve to polarize a single molecule
of water making it more nucleophilic towards the peptide bond to be cleaved.
The hydrolysis of PTMS by these three enzymes was followed by
29
Si NMR
spectroscopy. While the conditions that were employed were not considered optimal
for any of these enzymes, it was important to minimize any background hydrolysis
rate, known to be slowest at neutral pH, [
5
] and to treat each enzyme with identical
processing parameters. The hypothesis was that there was a correlation between the
architecture of the
S1
binding pocket and the rate of hydrolysis of PTMS.
The integration values from each
29
Si NMR spectrum can be used to follow the
hydrolysis of PTMS. A plot of ln[
29
Si] versus time results in a straight line, the slope
of which is the observed pseudo first order rate constant [
23
,
31
]. The pseudo first
order rate constants derived from these experiments are summarized in Table
3.2
and Fig.
3.4
.
Increasing the enzyme concentration was followed by an increase in the rate con-
stant (Fig.
3.4
) [
32
]. Trypsin was the least proficient at hydrolyzing PTMS. Further-
more, trypsin also showed the smallest changes in the rate constant as the amount of
the enzyme was increased. On the other hand pepsin displayed the highest rates of
hydrolysis, and like trypsin, exhibited only small changes in the rate constant as the
concentration was increased. If the line representing pepsin was to be extrapolated
to lower concentrations it would not pass through the origin. Unfortunately we can-
not make any comment on the behaviour of the enzyme at those concentrations as
a result of difficulties in obtaining reliable
29
Si NMR data. The rate of hydrolysis
of PTMS by α-chymotrypsin at lower concentrations was intermediate compared to
trypsin and pepsin, but as the amount α-chymotrypsin was increased, the associated
change in the rate constant was the largest of the three enzymes.
