1KEC: Penicillin Acylase Mutant With Phenyl Proprionic Acid

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.
PDB ID: 1KECDownload
MMDB ID: 23851
PDB Deposition Date: 2001/11/15
Updated in MMDB: 2012/12
Experimental Method:
x-ray diffraction
Resolution: 2.3  Å
Source Organism:
Similar Structures:
Biological Unit for 1KEC: dimeric; determined by author and by software (PISA)
Molecular Components in 1KEC
Label Count Molecule
Proteins (2 molecules)
Penicillin Acylase Alpha Subunit
Molecule annotation
Penicillin Acylase Beta Subunit
Molecule annotation
Chemicals (2 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB