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Title
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Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.
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Authors
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P.Strop,
J.Sedlacek,
J.Stys,
Z.Kaderabkova,
I.Blaha,
L.Pavlickova,
J.Pohl,
M.Fabry,
V.Kostka,
M.Newman.
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Ref.
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Biochemistry, 1990,
29,
9863-9871.
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PubMed id
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Abstract
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Comparison of the three-dimensional structure of bovine chymosin with the
structures of homologous aspartic proteinases complexed with peptide inhibitors
shows that Val111 in chymosin occupies a position between the specificity
subsites S1 and S3. A mutation corresponding to Val111 to Phe has been
introduced in an intermediary plasmid construct of prochymosin by bridging its
unique restriction sites by a synthetic mutant oligonucleotide duplex. A
prochymosin fusion product was expressed in Escherichia coli in such a way that
the extension and substitution of the propart does not interfere with the
activation of the zymogen. After activation of the crude prochymosin, the enzyme
was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as
ligand. This procedure provided large amounts of pure protein as judged by FPLC,
the activity/protein ratio, and SDS-PAGE. The enzymatic properties were
determined by using a variety of peptide substrates and inhibitors; KM values
for the mutant enzyme were approximately twice those of the wild type, but the
kcat values were little changed. The mutant enzyme was crystallized, X-ray data
were collected to 2.0-A resolution by using a FAST area detector, and the
structure was solved by using difference Fourier methods and refined to an R
factor of 19.5%. The mutation leads to only local changes in conformation, with
the phenylalanine side chain occupying part of the S1 and S3 pockets. This
accounts for the increased KM of this mutant for a substrate with a large
phenylalanine side chain at P1. It is also consistent with the higher affinity
of the mutant for an inhibitor with small side chains at P1 and P3 when compared
with the wild-type enzyme.
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