PENICILLIN ACYLASE HAS A SINGLE-AMINO-ACID CATALYTIC CENTER

PENICILLIN acylase (penicillin amidohydrolase, EC3.5.1.11) is widely d istributed among microorganisms, including bacteria, yeast and filamen tous fungi. It is used on an industrial scale for the production of 6- aminopenicillanic acid, the starting material for the synthesis of sem i-synthetic penicillins. Its in vivo role remains unclear, however, an d the observation that expression of the Escherichia coli enzyme in vi vo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mod e(1). The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively(2,3)) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal se quence to direct export to the cytoplasm and a 54-amino-acid spacer be tween the A and B chains which may influence the final folding of the chains(5). The N-terminal serine of the B chain reacts with phenylmeth ylsulphonyl fluoride, which is consistent with a catalytic role for th e serine hydrouyl group. Modifying this serine to a cysteine(6,7) inac tivates the enzyme, whereas threonine, arginine or glycine substitutio n prevents ill vivo processing of the enzyme indicating that this must be an important recognition site for cleavage. Here,ve report the cry stal structure of penicillin acylase at 1.9 Angstrom resolution. Our a nalysis shows that the environment of the catalytically active N-termi nal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own alpha-amino group, which may act by a new me chanism to endow the enzyme with its catalytic properties.