Figure 4.
Fig. 4. Catalytic mechanism for DERA. (A) 1H NMR experiment.
DERA-catalyzed exchange of C2 proton of
(R)-2-deuteropropanaldehyde and (S)-2-deuteropropanaldehyde in
D[2]O was established by 1H NMR. Incubation of
(R)-2-deuteropropanaldehyde with DERA in D[2]O effects complete
exchange of the -proton to
a deuteron, as observed by the collapse of the C3 doublet
resonances of the aldehyde and acetal to singlets (left). By
contrast, incubation of (S)-2-deuteropropanaldehyde with DERA in
D[2]O does not effect proton exchange, as observed by retention
of the doublet resonances (right). (B) Proposed catalytic
mechanism for DERA. The proposed mechanism is consistent with
all of our ultra-high resolution structural, modeling,
site-directed mutagenesis, and 1H NMR data. Lys167 is identified
as the Schiff base-forming residue. After the enamine is formed,
the system is poised for nucleophilic attack onto the
carbonyl-carbon of the acceptor aldehyde
D-glyceraldehyde-3-phosphate. A proton relay system between
Asp102, Lys201, and an active site water molecule is responsible
for shuffling a proton between C2 of the acetaldehyde imine and
enamine and subsequent C3 hydroxyl protonation. The double
arrows in green indicate rapid proton shuffling between Lys201
and Asp102. Crystallographically observed reaction intermediates
are boxed (left, carbinolamine; right, Schiff base). (C)
Stereochemical course of aldol reaction. When propanal is
substituted for acetaldehyde as the donor, the pro-S proton is
removed, and thus the aldol reaction proceeds with retention of
configuration at C2, with the Si face of the resulting enamine
approaching the Re face of the acceptor carbonyl. A:H, general
acid; B, general base.
The above figure is reprinted
by permission from the AAAs:
Science
(2001,
294,
369-374)
copyright 2001.
-proton to
a deuteron, as observed by the collapse of the C3 doublet
resonances of the aldehyde and acetal to singlets (left). By
contrast, incubation of (S)-2-deuteropropanaldehyde with DERA in
D[2]O does not effect proton exchange, as observed by retention
of the doublet resonances (right). (B) Proposed catalytic
mechanism for DERA. The proposed mechanism is consistent with
all of our ultra-high resolution structural, modeling,
site-directed mutagenesis, and 1H NMR data. Lys167 is identified
as the Schiff base-forming residue. After the enamine is formed,
the system is poised for nucleophilic attack onto the
carbonyl-carbon of the acceptor aldehyde
D-glyceraldehyde-3-phosphate. A proton relay system between
Asp102, Lys201, and an active site water molecule is responsible
for shuffling a proton between C2 of the acetaldehyde imine and
enamine and subsequent C3 hydroxyl protonation. The double
arrows in green indicate rapid proton shuffling between Lys201
and Asp102. Crystallographically observed reaction intermediates
are boxed (left, carbinolamine; right, Schiff base). (C)
Stereochemical course of aldol reaction. When propanal is
substituted for acetaldehyde as the donor, the pro-S