Biomedical Engineering Reference
In-Depth Information
a schematic representation of the canonical reaction cycle of an aspartyl protease, illustrating the
changes in active site structure that attend catalysis. Before substrate binding the enzyme is in a
resting form (E) in which the two active site aspartic acid residues are bridged by a water mol-
ecule. One of the aspartates is present in the protonated acid form while the other is present as the
conjugate base form, and the two residues share the acid proton through a strong hydrogen bond.
Initial substrate binding causes disruption of the hydrogen bonding interactions and displacement
of the water molecule in species ES. After initial substrate binding, a l exible loop of the protein,
referred to as the “l ap” closes down over the active site to occlude the active site groups from bulk
solvent. The substrate-bound enzyme in this altered conformation is referred to as form E
S in
Figure 11.1. Subsequently, the water of the enzyme active site attacks the carbonyl carbon of the
scissile peptide bond to form a dioxy, tetrahedral carbon center on the substrate. This constitutes
the enzyme-bound transition state of the reaction and is symbolized as E
′
S
‡
in the i gure. Bond
rupture then occurs, leading to a species with both active site aspartates protonated and with both
the anionic and cationic peptide products bound (form E
′
P). After that, the l ap retracts from the
active site to generate a new conformational state of the active site, referred to as FP. With the l ap
out of the way, the product peptides can now dissociate from the enzyme, forming enzyme state
F. Deprotonation of one of the active site aspartates occurs next to form state G. Finally, addition
of a water molecule returns the enzyme back to the original conformational state E, thus complet-
ing the catalytic cycle.
It is clear from Figure 11.1 that protein dynamics is an important component of the catalytic
cycles of enzyme reactions. As stated earlier, the importance of this concept to drug discovery is
that each intermediate state accessed along the reaction pathway provides unique opportunities for
inhibitor interactions. For example, in the case of the aspartyl proteases, there are three distinct
ligand-free conformational forms of the enzyme (states E, F, and G); small molecule inhibitors are
known that preferentially bind to each of these individual states.
The protein structures that are represented along the reaction pathway of an enzyme rel ect
a collection of conformational microstates that interconvert among themselves through rotational
and vibrational excursions. Thus, any particular conformation of the enzyme can be represented as
a manifold of conformational substates (microstates), each stabilized to different degrees by spe-
cii c interactions with ligands. Isomerization of the enzyme from one structure to another therefore
involves the potential energy stabilization of certain microstates at the expense of others.
Similarly, high-afi nity inhibitor interactions often involve isomerization of the enzyme from
an initial structure resembling the unliganded enzyme to a new conformation in which a particular
microstate(s) is highly stabilized by interactions with the inhibitor. Kinetically, this requires two
distinct steps in overall binding of high-afi nity inhibitors. As illustrated in Figure 11.2A, the i rst
step involves the formation of a reversible encounter complex between the enzyme and inhibitor (EI),
which often displays only modest afi nity. Forward binding of the inhibitor to the enzyme is dic-
tated by the association rate constant
k
3
and dissociation of the initial EI complex is dictated by
the dissociation rate constant
k
4
. Once the EI complex is formed, enzyme isomerization can occur
to form the much higher afi nity complex E*I. The forward conversion of EI to E*I is dictated by
the isomerization rate constant
k
5
and the reverse conversion of E*I back to EI is dictated by rate
constant
k
6
. All three enzyme forms (E, EI, and E*I) can also be represented as potential energy
diagrams, as illustrated in Figure 11.2B. It should be noted that the afi nity of the enzyme-inhibitor
complex is related to the potential energy stabilization of the system, which is rel ected in the depth
of the potential well in the energy diagram. The deeper the potential energy well for the inhibited
form, the more energy that is required to escape this well and thus access the other conformational
microstates required for continued catalysis. The potential energy stabilization of the inhibitor-bound
form is mediated by productive interactions between the inhibitory ligand and the binding pocket of
the protein, in the form of hydrogen bonds, electrostatic interactions, hydrophobic interactions, van
der Waal forces, and the like. These concepts can also be conceptualized in terms of an induced i t
between the binding pocket and the inhibitor, as illustrated in cartoon form in Figure 11.2C.
′
