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Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of b-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of aF146, bF24 and bF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions aF146 and bF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for aF146Y and aF146W to a threefold increase for aF146L and bF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the bF24A mutant showed that it had specificity constants (k cat /K m ) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased k cat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the k cat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of bF24A resulted in high yields in acyl transfer reactions.Keywords: site-directed mutagenesis; b-lactam antibiotics; penicillin acylase; substrate specificity; transferase/ hydrolase ratio.Penicillin acylase (PA) of Escherichia coli (EC 3.5.1.11) catalyses the hydrolysis of penicillin G to phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). PA is a heterodimeric periplasmic protein consisting of a small a subunit and a large b subunit, which are formed by processing of a precursor protein. The catalytic nucleophile, a serine, is located at the N-terminus, which is a hallmark of the family of N-terminal nucleophile (Ntn) hydrolases, a class of enzymes which share a common fold around the active site and contain a catalytic serine, cysteine or threonine at the N-terminal position [1]. The reaction mechanism of PA involves the formation of a covalent intermediate and is similar to the well-known mechanism of serine proteases. After attack on the carbonyl carbon of the amide bond by the active-site nucleophile, a covalent acyl-enzyme is formed via a tetrahedral transition state in which the negatively charged oxyanion is stabilized by H-bonds to the oxyanion hole residues bN241 and bA69 [2]. After expulsion of the leaving group from the active site, the acyl-enzyme is deacylated by H 2 O or another nucleophile, yielding the final transacylation product and the free enzyme.PA is used for the production of 6-aminopenicillanic acid (6-APA) by the hydrolysis of penicillin G, but can also be used for the production of semisynthetic b-lactam antibiotics, in which the enzyme catalyses the condensation of an acyl group and a 6-APA molecule [3]. In this cond...

Atrial fibrillation (AF) is the most common sustained clinical tachyarrhythmia. AF is a progressive condition as demonstrated by the finding that maintenance of normal rhythm and contractile function becomes more difficult the longer AF exists. AF causes cellular stress, which induces atrial remodelling, involving reduction in the expression of L-type Ca(2+) channels and structural changes (myolysis), finally resulting in contractile dysfunction. Heat shock proteins (HSPs) comprise a family of proteins involved in the protection against different forms of cellular stress. Their classical function is the prevention of toxic protein aggregation by binding to (partially) unfolded proteins. Recent investigations reveal that HSPs prevent atrial remodelling and attenuate the promotion of AF in both cellular and animal experimental models. Furthermore, studies in humans suggest a protective role for HSPs against progression from paroxysmal AF to chronic, persistent AF. Therefore, manipulation of the HSP system may offer novel therapeutic approaches for the prevention of atrial remodelling. Such approaches may contribute to the maintenance or restoration of tissue integrity and contractile function. Ultimately, this concept may offer an additional treatment strategy to delay progression towards chronic AF and/or improve the outcome of cardioversion.