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Title
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Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase.
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Authors
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A.T.Danishefsky,
J.J.Onnufer,
G.A.Petsko,
D.Ringe.
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Ref.
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Biochemistry, 1991,
30,
1980-1985.
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PubMed id
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Abstract
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Arginine-386, the active-site residue of Escherichia coli aspartate
aminotransferase (EC 2.6.1.1) that binds the substrate alpha-carboxylate, was
replaced with tyrosine and phenylalanine by site-directed mutagenesis. This
experiment was undertaken to elucidate the roles of particular enzyme-substrate
interactions in triggering the substrate-induced conformational change in the
enzyme. The activity and crystal structure of the resulting mutants were
examined. The apparent second-order rate constants of both of these mutants are
reduced by more than 5 orders of magnitude as compared to that of wild-type
enzyme, though R386Y is slightly more active than R386F. The 2.5-A resolution
structure of R386F in its native state was determined by using difference
Fourier methods. The overall structure is very similar to that of the wild-type
enzyme in the open conformation. The position of the Phe-386 side chain,
however, appears to shift with respect to that of Arg-386 in the wild-type
enzyme and to form new contacts with neighboring residues.
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