Ligand-induced Conformational Change in Penicillin Acylase Sarah H. Done*, James A. Brannigan, Peter C. E. Moody and Roderick E. Hubbard Department of Chemistry University of York, Heslington York YO1 5DD, UK The enzyme penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) cat- alyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the pro- duct 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. Studies have shown that the enzyme is speci®c for hydrolysis of phenylacetamide derivatives, but is more tolerant of fea- tures in the rest of the substrate. It is this property that has led to many other applications for the enzyme, and greater knowledge of the enzyme's structure and speci®city could facilitate engineering of the enzyme, enhancing its potential for chemical and industrial applications. An extensive study of the binding of a series of phenylacetic acid derivatives has been carried out. A measure of the relative degree of inhi- bition of the enzyme by each of the compounds has been obtained using a competitive inhibition assay, and the structures of a number of these complexes have been determined by X-ray crystallography. The struc- tures reveal a clear rationale for the observed kinetic results, but show also that some of the ligands cause a conformational change within the binding pocket. This change can generally be understood in terms of the size and orientation of the ligand within the active site. The results reveal that ligand binding in penicillin acylase is facilitated by certain amino acid residues that can adopt two distinct, energetically favourable positions in order to accommodate a variety of compounds within the active site. The structures of these complexes provide evidence for conformational changes in the substrate-binding region that may act as a switch in the mechanism of autocatalytic processing of this enzyme. # 1998 Academic Press Keywords: penicillin acylase; phenylacetic acid; ligand binding; conformational change; crystallography *Corresponding author Introduction Penicillin G acylase from Escherichia coli W is produced as a single-chain precursor consisting of 846 amino acid residues in the cytoplasm. The pre- cursor is then processed by the removal of a 26 residue signal peptide and a 54 residue spacer pep- tide, to produce a mature enzyme in the periplasm that is a heterodimer of A and B chains of 209 and 557 amino acid residues, respectively (Bru È ns et al., 1985). The processing of the spacer peptide has been studied by mutational analysis and is thought to be autocatalytic (Choi et al., 1992; Schumacher et al., 1986; Sizmann et al., 1990). Although the natural function of the enzyme is as yet unclear, it has been suggested that penicillin acylase is involved in the degradation of phenylacetylated compounds for the generation of phenylacetic acid, which may be used as a carbon source when the bacterium is in its free-living mode (Valle et al., 1991). It has also been noted that the penicillin acy- lase gene in Escherichia coli is located near a gene cluster encoding the 4-hydroxyphenylacetic acid degradative pathway (Prieto et al., 1993, 1996). This means that penicillin acylase could be involved in E-mail address of the corresponding author: done@yorvic.york.ac.uk Present address: P. C. E. Moody. Department of Biochemistry, Adrian Building, University of Leicester, University Road, Leicester LE1 7RH, UK. Abbreviation used: Ntn, N-terminal nucleophile. Article No. mb982180 J. Mol. Biol. (1998) 284, 463±475 0022 ± 2836/98/470463±13 $30.00/0 # 1998 Academic Press