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Title
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Structure and dynamics of the metal site of cadmium-substituted carboxypeptidase A in solution and crystalline states and under steady-state peptide hydrolysis.
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Authors
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R.Bauer,
E.Danielsen,
L.Hemmingsen,
M.V.Sorensen,
J.Ulstrup,
E.P.Friis,
D.S.Auld,
M.J.Bjerrum.
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Ref.
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Biochemistry, 1997,
36,
11514-11524.
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PubMed id
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Abstract
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PAC spectra (perturbed angular correlation of gamma-rays) of cadmium-substituted
carboxypeptidase A (CPD) show that the enzyme in solution imposes a flexible,
pH- and chloride-dependent coordination structure on the metal site, in contrast
to what is found in the crystalline state. A much more restricted coordination
geometry occurs for the steady-state peptide intermediates of Bz-Gly-l-Phe and
Bz-Gly-Gly-l-Phe in solution, suggesting that substrate binding locks the
structure in a rigid conformation. The results further indicate that the peptide
intermediate has a six-coordinated metal coordination geometry with an OH-
ligand at the solvent site and a carbonyl oxygen at an additional ligand site.
In marked contrast, conformational rigidity is not induced by the inhibitor/poor
substrate Gly-L-Tyr nor by the products of high turnover substrates, Bz-Gly,
Bz-Gly-Gly, and L-Phe. These results are consistent with an intact scissile
peptide bond in the enzyme-substrate complex of Bz-Gly-L-Phe and
Bz-Gly-Gly-L-Phe. A single nuclear quadrupole interaction (NQI) is observed for
the crystalline state of the enzyme between pH 5.7 and pH 9.4. This NQI agrees
with calculations based on the metal coordination geometry for cadmium in
crystalline CPD derived from X-ray diffraction studies. A single broad
distribution of NQIs is observed for CPD in sucrose solutions and 0.1 M NaCl at
pH values below 6.5. This NQI (NQI-1') has parameters very close to those for
the crystalline state. The enzyme metal site, characterized by this NQI, is
converted into two new enzyme metal sites over the pH range of 6.5-8.3. The
metal coordination sphere of one of these has a NQI (NQI-1) with parameters
similar to those at lower pH values (NQI-1') while the other NQI (NQI-2) is
characterized by markedly different NQI parameters. Angular overlap model (AOM)
calculations indicate that the coordination sites giving NQI-1' and NQI-1 both
have a metal-bound water molecule while the coordination site giving NQI-2 has a
metal-bound hydroxide ion. PAC results at pH 8.3-10.5 indicate that in this pH
range the two metal coordination geometries related to NQI-1 and NQI-2 occur in
a pH independent ratio of 2:1, with the one with the water ligand being the most
abundant species. The observed pH-independent equilibrium between the two
different metal coordination geometries for cadmium can be explained by an
equilibrium between tautomeric forms of a hydrogen bond between the Glu-270
carboxyl group and the metal-bound water (Glu-270 COO-...(HOH)M <==>
Glu-270 COOH...(OH-)M) being slow on the time scale of a PAC experiment, i.e.,
slower than 0.5 micros. We finally suggest that NQI-1' observed at low pH
reflects an enzyme species containing a metal-coordinated water molecule and the
protonated carboxyl group of Glu-270.
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