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Enzymatic Hydrolysis and Group Transfer Reactions 97
HO
HO
HO
ENZ
peptide-NH
O
N
R
H 2
peptide-NH
H
N
OH
R
peptide-NH
N
R
hydrolysis products
Figure 5.18 18 O exchange via a hydrate intermediate.
observed at up to 10% of the rate of the forward reaction. Since the reaction is
in practice irreversible, this exchange is consistent with the reversible formation
of a hydrated intermediate, as shown in Figure 5.18.
D 2 O solvent isotope eVects of 1.5–3.2 have been measured under a range of
conditions, consistent with base-catalysed attack of water being the Wrst step
of the mechanism. Finally, small inverse 15 N isotope eVects have been measured
for the departing nitrogen atom, suggesting protonation of nitrogen in the ratedetermining
step of the mechanism. The proposed mechanism is illustrated in
Figure 5.19.
According to this mechanism Asp-25 acts as a base to deprotonate an
attacking water molecule, with Asp-25 0 acting as a general acid, forming the
hydrated intermediate. Breakdown of this intermediate with protonation of
the departing nitrogen atom by the protonated Asp-25 completes the catalytic
cycle.
Most of the research eVort on the HIV-1 protease has been directed towards
synthesising potent inhibitors. Given the precedented inhibition of pepsin and
other aspartyl proteases by pepstatin, a range of substrate analogues containing
statine-like groups have been synthesised and found to act as potent inhibitors
for the HIV-1 protease. It is thought that the statine unit mimics the tetrahedral
intermediate formed in the reaction, as shown in Figure 5.20.
Administration of human T-lymphocyte cells that are infected with the
HIV-1 virus with such inhibitors has demonstrated that these compounds
HO
HO
HO
peptide-NH
H
O
O
Asp ' 25
O
N
O R
H
H
− O O
Asp 25
peptide-NH
H
O − O
Asp ' 25
N
O OH
H
O
R
O
Asp 25
peptide-NH
H
O
O
Asp ' 25
O
OH
− O
HN
R
O
Asp 25
Figure 5.19 Mechanism for HIV-1 protease.