Comparison of the active site of pro-TPP1 zymogen with that of mature
kumamolisin. Backbones are shown as ribbon representations with
featured side chains in ball-and-stick mode, bound water molecules
depicted as red spheres, and hydrogen bonds shown as dashed
lines with distances in Å. A, stereo view of TPP1 showing
catalytic residues in a conformation that is recalcitrant to activity. In
TPP1, Glu272 (E), Asp276 (D), and
Ser475 (S) form the catalytic triad, and Asp360
(D′) forms part of the stabilizing oxyanion hole.
Ser324 (S′) is completely conserved in the S53
family. Backbone carbonyl groups are shown only for the Ser and S′
residues. Side chain oxygen atoms are shown in red and backbone
carbonyl oxygens in yellow. Both Glu272 and
Asp276 side chains are oriented away from Ser475. The
position of Glu272 is stabilized by a hydrogen bond to
Gln233. A bound water molecule (1) occupies the position
expected for one of the carboxyl oxygens of Glu272, if the active
site were poised for activity. One of the carboxyl oxygens of
Asp276 forms a hydrogen bond to a water molecule (2), and
the other is within weak hydrogen bond distance to the sulfhydryl of unpaired
Cys277. The side chain hydroxyls of Ser475 and
Ser324 participate in a strong hydrogen bond. A third water
molecule (3) makes a strong hydrogen bond with the Ser475
hydroxyl and is also within hydrogen bond distance of the Asp360
carboxyl constituting the oxyanion hole, close to where the carbonyl oxygen of
the scissile peptide bond is expected to be positioned. B, comparison
of catalytic machinery of pro-TPP1 with that in mature kumamolisin. Least
square superpositions of Cα coordinates of the pro-TPP1
catalytic domain with those of all available structures of S53 family members
were performed and yielded similar results. Superposition of TPP1 with mature
kumamolisin (PDB ID 1GTL) (27)
is shown with ribbon depictions in gray and green,
respectively. Although the corresponding oxyanion D′ residues in TPP1
and kumamolisin are in similar conformations, the corresponding catalytic Glu
and Asp residues are both in different conformations. Furthermore, the
S′ residue in TPP1 (Ser324) is in a different rotameric
conformation, and its Cα position is displaced by 1.5 Å
from the corresponding S′ (Ser316) in kumamolisin, resulting
in different hydrogen bond schemes with the active site Ser.