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Aspartate-ß-Semialdehyde
Dehydrogenase
Background:
ASA DH exists as
a homodimer, with each subunit containing 367 amino acids and a subunit
molecular weight of 39,950. Chemical modification and pH profile studies
have suggested the presence of a single cysteine at the active site of ASA
DH (Karsten & Viola, 1991).
We have probed this enzyme by site-directed mutagenesis to identify
residues that play an important function in catalytic activity.
Cysteine-135 has been shown to be the active site nucleophile (Karsten & Viola, 1992),
substrate binding is facilitated by an arginine, and residues involved in
coenzyme binding have been identified (Ouyang & Viola, 1995).
Structure:
The high
resolution structure of ASA DH has been determined both as the apo-enzyme and as enzyme-substrate complexes. The
intimate relationship between the monomers clearly identifies this enzyme
as a biologically active dimer. There are two distinct domains in each monomer,
the N-terminal domain containing a classic NADP-binding fold, and a second
domain containing an -helix surrounded by a six-stranded ß-sheet
which is at the subunit interface. The active site contains the cys-135
nucleophile in close contact with his-274, which then appears to be
hydrogen-bonded to gln-162. Arg-267 is involved in binding to the substrate
carboxyl group. Work in progress involves stabilizing and solving the
structure of the acyl- enzyme intermediate in the catalytic cycle.
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