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PDBsum entry 1kec
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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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Structural and kinetic studies on ligand binding in wild-Type and active-Site mutants of penicillin acylase.
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Authors
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W.B.Alkema,
C.M.Hensgens,
H.J.Snijder,
E.Keizer,
B.W.Dijkstra,
D.B.Janssen.
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Ref.
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Protein Eng Des Sel, 2004,
17,
473-480.
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PubMed id
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Abstract
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Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic
acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam
antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a
high affinity for Calpha-substituted phenylacetic acid derivatives is desirable.
We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A,
which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds
than wild-type enzyme. In addition, betaF24A had a 20-fold reduced affinity for
phenylacetic acid. The molecular basis of the improved properties was
investigated by X-ray crystallography. These studies showed that the higher
affinity of alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by
van der Waals interactions between alphaY146:OH and the Calpha-substituent. The
betaF24A mutation causes an opening of the phenylacetic acid binding site. Only
(R)-alpha-methylphenylacetic acid, but not phenylacetic acid, induces a
conformation with the ligand tightly bound, explaining the weak binding of
phenylacetic acid. A comparison of the betaF24A structure with other open
conformations of penicillin acylase showed that betaF24 has a fixed position,
whereas alphaF146 acts as a flexible lid on the binding site and reorients its
position to achieve optimal substrate binding.
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