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PDBsum entry 1sio
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Hydrolase/hydrolase inhibitor
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PDB id
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1sio
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Crystallographic and biochemical investigations of kumamolisin-As, A serine-Carboxyl peptidase with collagenase activity.
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Authors
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A.Wlodawer,
M.Li,
A.Gustchina,
N.Tsuruoka,
M.Ashida,
H.Minakata,
H.Oyama,
K.Oda,
T.Nishino,
T.Nakayama.
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Ref.
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J Biol Chem, 2004,
279,
21500-21510.
[DOI no: ]
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PubMed id
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Abstract
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Kumamolisin-As (previously called ScpA) is the first known example of a
collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low
pH and in elevated temperatures. In this study that used x-ray crystallographic
and biochemical methods, we investigated the structural basis of the preference
of this enzyme for collagen and the importance of a glutamate residue in the
unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity.
Crystal structures of the uninhibited enzyme and its complex with a covalently
bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence
of a narrow S2 pocket and a groove that encompasses the active site and is rich
in negative charges. Limited endoproteolysis studies of bovine type-I collagen
as well as kinetic studies using peptide libraries randomized at P1 and P1',
showed very strong preference for arginine at the P1 position, which correlated
very well with the presence of a negatively charged residue in the S1 pocket of
the enzyme. All of these features, together with those predicted through
comparisons with fiddler crab collagenase, a serine peptidase, rationalize the
enzyme's preference for collagen. A comparison of the Arrhenius plots of the
activities of kumamolisin-As with either collagen or peptides as substrates
suggests that collagen should be relaxed before proteolysis can occur. The E78H
mutant, in which the catalytic triad was engineered to resemble that of
subtilisin, showed only 0.01% activity of the wild-type enzyme, and its
structure revealed that Ser(278), His(78), and Asp(82) do not interact with each
other; thus, the canonical catalytic triad is disrupted.
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Figure 5.
FIG. 5. A superposition of the active sites of four
proteases. Kumamolisin-As with bound AcIPF is shown in blue;
sedolisin and bound tyrostatin are colored red and brown,
respectively; subtilisin is shown in lilac, and its inhibitor
eglin is shown in violet. Fiddler crab collagenase is colored
green, whereas its inhibitor ecotin is orange. The C  atoms of
kumamolisin-As, sedolisin, and subtilisin were superimposed with
the program ALIGN (34). Superposition of kumamolisin-As with
fiddler crab collagenase is described under "Results."
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Figure 6.
FIG. 6. Superposition of kumamolisin-As and fiddler crab
collagenase. The two structures were superimposed as described
under "Results." The main chain tracing of kumamolisin-As and
fiddler crab collagenase is shown as red and blue ribbons,
respectively, and the superimposed side chains of the active
sites residues are shown as sticks in corresponding colors. The
strands 127-132 in kumamolisin-As and 213-218 in collagenase are
emphasized in black (see under "Results"). The inhibitor AcIPF
is shown in gold, and a fragment of ecotin is shown in green.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
21500-21510)
copyright 2004.
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