|
The structure of the complex of L-benzylsuccinate (Ki = 0.2 mM) bound to wheat
serine carboxypeptidase II has been analyzed at 2.0-A resolution for native and
inhibited crystals at -170 degrees C. The model has been refined and has a
standard crystallographic R-factor of 0.176 for 57,734 reflections observed
between 20.0- and 2.0-A resolution. The root mean square deviation from ideal
bonds is 0.017 A and from ideal angles is 2.6 degrees. The model consists of 400
amino acids, 4 N-linked saccharide residues, and 430 water molecules.
L-Benzylsuccinate occupies a narrow slot in the active site defined by Tyr 60,
Tyr 239, and the polypeptide backbone. One carboxylate forms hydrogen bonds to
Glu 145, Asn 51, the amide of Gly 52, and the catalytic His 397, suggestive of
how the peptide C-terminal carboxylate is recognized by the enzyme. The phenyl
ring stacks between Tyr 239 and Tyr 60, while the other carboxylate occupies the
"oxyanion hole". One of the oxygens accepts hydrogen bonds from the amides of
Tyr 147 and Gly 53, while the other forms a very close contact (2.3 A) with the
O gamma of Ser 146, forcing the side chain into a conformation alternative to
that found in the resting state of the enzyme. The inhibitor occupies the active
site in a way that suggests that it can be regarded as a transition-state
analogue of serine carboxypeptidases. The model suggests a novel enzymatic
mechanism, involving substrate-assisted catalysis, that might account for the
low pH optimum (4.0-5.5) of peptidase activity unique to this family of serine
proteinases.
|
