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PDBsum entry 1ggd
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Hydrolase/hydrolase inhibitor
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PDB id
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1ggd
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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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Correlation of low-Barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin.
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Authors
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D.Neidhart,
Y.Wei,
C.Cassidy,
J.Lin,
W.W.Cleland,
P.A.Frey.
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Ref.
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Biochemistry, 2001,
40,
2439-2447.
[DOI no: ]
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PubMed id
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Abstract
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The structures of the hemiketal adducts of Ser 195 in chymotrypsin with
N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and
N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to
1.4-1.5 A by X-ray crystallography. The structures confirm those previously
reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990)
Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and
Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the
low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the
basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure
of the hemiacetal adduct between Ser 195 of chymotrypsin and
N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0.
The structure is similar to the AcLF-CF3 adduct, except for the presence of two
epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In
the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site.
On the basis of the downfield 1H NMR spectrum in solution, His 57 is not
protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of
AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution,
it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex
is a hydroxyl group and not attracted to the oxyanion site. The protonation
states of the hemiacetal and His 57 are explained by the high basicity of the
hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR
signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a
neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the
AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2
of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm.
The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an
energy barrier of 6-7 kcal x mol(-1) against ionizations that change the
electrostatic charge at the active site. However, ionizations of neutral His 57
in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no
apparent barrier. Protonation of His 57 is accompanied by LBHB formation,
suggesting that stabilization by the LBHB overcomes the barrier to ionization.
On the basis of the hydration constant for AcLF-13CHO and its inhibition
constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for
AcLF-CF3.
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