Download Extra slides (lecture Wed. 11/4)

Binding features that promote catalysis
• co-localization and proper orientation of reacting
groups
• involving different molecules or just a single
molecule
• binding energy is used to pay the entropic cost of
bringing reacting groups into the right positions
and orientations
• stabilization of (and thereby population of) particular
configurations of molecules suitable for bond
formation/breakage
• electrostatic contributions; stabilization of the charged
states that develop during a reaction
Figure 11-13b
An example of the importance of proximity
effects
slow reaction
fast reaction
Figure 11-13b
Understanding catalysis as a lowering of
the transition state free energy
• articulated by Linus Pauling
• recall that providing something that binds a molecule
tightly effectively lowers its free energy
If the enzymes binding site
is most complementary to
the transition state (and so
lowers the energy of the
transition state more than
the substrate/products),
then the activation free
energy is lowered, leading
to rate enhancement.
Figure 11-13b
The importance of transition state theory
• helps explain the mechanisms of catalysis
• provides a strategy for designing inhibitors against
various enzymes
• imagine what the (unstable) t.s. for the reaction must
look like
• design a (stable) organic mimic that looks similar; this
should bind very more tightly to the enzyme than the
substrate, and act as a potent inhibitor
• an important strategy in drug design
Page 339
Example of an inhibitor based on a
transition state analogue
reaction catalyzed
by the enzyme
proline racemace
transition state
analogues sharing
some features
with the presumed
transition state
Page 339
Mechanism of a well-studied enzyme: lysozyme
• hydrolyzes b(14) polysaccharides linkages in the
bacterial cell wall
• resembles an acid catalyzed mechanism in solution
• Glu 35 acts as an acid, donating proton to leaving group
• carbocation stabilized by
• oxonium resonance, promoted by binding the sugar
ring in a half-chair configuration
• then by covalent bond to Asp 52
• 2nd half of the reaction is the reverse of the first
• H20 displaces the leaving sugar
• Glu 35 now acts as a base to accept proton from
H20, which becomes the attacking group when the
Asp 52 leaves
• note that ‘double displacement’ leads to retention of the
b configuration at the anomeric carbon
Page 339
substrate
enzyme. note the
active site has subsites for binding
multiple parts of
the sustrate
Figure 11-16
A planar arrangement of
atoms attached to the ring
oxygen is required for
stabilization of the
carbocation that develops
at the anomeric carbon
when the leaving group
leaves. Such a planar
configuration is provided
by the half-chair ring
conformation, which is
how the active site binds
the substrate.
Figure 11-19
Lysozyme mechanism
Figure 11-21
Lysozyme mechanism
Figure 11-21 part 1
Lysozyme mechanism
Figure 11-21 part 2
Lysozyme mechanism
Figure 11-21 part 3
Lysozyme mechanism
Figure 11-21 part 4
Lysozyme mechanism
Figure 11-21 part 5
Lysozyme transition state and t.s. analog
Figure 11-22
Mechanism of a well-studied family of enzymes:
serine proteases
• proteases hydrolyze other
proteins
• widespread
• use a serine nucleophile to
attack substrate carbonyl
• because the serine
nucleophiles in these
enzymes are so
nucleophilic, they can be
‘killed’ (i.e. irreversibly
modified by a covalent
bond) by ‘suicide
inhibitors’
Page 339
Mechanism of a well-studied family of enzymes:
serine proteases
• specificities of different serine
proteases vary (e.g. trypsin,
chymotrypsin, etc.)
• specificity pockets in the
binding site for recognizing
certain amino acids in the
substrate proteins
Page 339
Mechanism of a well-studied family of enzymes:
serine proteases
• Mechanism:
• serine is promoted as a nucleophile by
deprotonation by nearby histidine side chain (whose
resulting positive charge is stabilized by a nearby
aspartate). This is the famous ‘catalytic triad’
• departure of C-terminal fragment of substrate
• a covalent intermediate is formed in which the Nterminal fragment of the substrate is attached to the
enzyme as an acyl-enzyme intermediate
• 2nd step is a direct repeat, but with H20 replacing the
serine as the nucleophile
Page 339
Serine proteases: the catalytic triad
note that the participating residues are coming
from different parts of the enzyme sequence
Figure 11-26
Similar catalytic triads have been discovered in apparently unrelated
enzymes, having different overall three-dimensional structures and
different orderings of the triad residues, suggesting that this
particularly useful arrangement of catalytic residues has evolved
independently multiple times (i.e. ‘convergent evolution’)
Figure 11-28
Serine protease mechanism
Figure 11-29
Serine protease mechanism
Figure 11-29 part 1
Serine protease mechanism
Figure 11-29 part 2
Serine protease mechanism
Figure 11-29 part 3
Serine protease mechanism
Figure 11-29 part 4
Serine protease mechanism
Figure 11-29 part 5
Serine proteases: features for transition state
stabilization
• ‘oxyanion hole’ to stabilize negative charge on
tetrahedral intermediate
• H-bonds to stabilize distorted protein backbone
Figure 11-30a
One biologically important use of proteases: to cleave
inactive proenzymes (or zymogens) to produce the
mature, active form of an enzyme
In some cases, such
cleavages act one after
another on a series of
enzymes. This leads to a
‘cascade’, which can
produce a geometric
explosion of enzymatic
activity (which may be
needed in response to
catastrophic events). The
well-known blood clotting
cascade is shown.
Box 11-4c