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PDBsum entry 1opy
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References listed in PDB file
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Key reference
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Title
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High-Resolution crystal structures of delta5-3-Ketosteroid isomerase with and without a reaction intermediate analogue.
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Authors
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S.W.Kim,
S.S.Cha,
H.S.Cho,
J.S.Kim,
N.C.Ha,
M.J.Cho,
S.Joo,
K.K.Kim,
K.Y.Choi,
B.H.Oh.
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Ref.
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Biochemistry, 1997,
36,
14030-14036.
[DOI no: ]
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PubMed id
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Abstract
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Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific
isomerization of steroid substrates at an extremely fast rate, overcoming a
large disparity of pKa values between a catalytic residue and its target. The
crystal structures of KSI from Pseudomonas putida and of the enzyme in complex
with equilenin, an analogue of the reaction intermediate, have been determined
at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side
chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen
bonds directly with the oxyanion of the bound inhibitor in a completely apolar
milieu of the active site. No water molecule is found at the active site, and
the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is
surrounded by six apolar residues, and consequently, its pKa appears to be
elevated as high as 9.5 to be consistent with early studies. No interaction was
found between the bound inhibitor and the residue 101 (phenylalanine in
Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to
contribute significantly to the rate enhancement based on mutational analysis.
This observation excludes the residue 101 as a potential catalytic residue and
requires that the rate enhancement should be explained solely by Tyr14 and
Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14
contributes much more significantly to the rate enhancement than Asp99. Previous
studies and the structural analysis strongly suggest that the low-barrier
hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength
hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required
energy of 11 kcal/mol for the transition-state stabilization.
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