Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

Abstract

Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F‐inactivated chymotrypsin [Gold, A. M. & Fahrney, D. (1964) Biochemistry 3, 783–791]. Incubation of the PhMeSO2F‐inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol‐enzyme during incubation with 10 mM 6‐aminopenicillanic acid. 4‐Pyridyl‐ethylcysteine was released by acid hydrolysis after reaction of the thiol‐protein with 4‐vinylpyridine. The rates of reaction of thiol‐penicillin acylase with iodoacetic acid and 2,2′‐dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol‐enzyme with iodo[2‐3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the β‐chain NH2‐terminal residue, indicating conversion of Ser290 to S‐carboxymethyl‐cysteine. Near‐ultraviolet CD spectra showed the conformation of thiol‐penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.

Structure, Processing and Catalytic Action of Penicillin Acylase

“…Recently, the 'chemical mutagenesis' reaction used to convert the subtilisin active site serine to cysteine (Neet and Koshland, 1966) has been applied to the enzymes from E. coil ATCC 11105 (Slade et al, 1990) and K. citrophila (Martin etal., 1990b). In each case, the 13-subunit N-terminal serine (Table 3, position 305) was converted to cysteine.…”

Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa

“…The catalytic residue, serine Ser-290, is located at the amino-terminus of the ␤-subunit (Daumy et al, 1985;Duggleby et al, 1995). Replacing this residue with cysteine results in a processed but inactive enzyme, associating the serine residue as a catalytic nucleophile (Choi et al, 1992;Slade et al, 1991). Moreover, substitution with arginine, threonine, or glycine impairs enzyme processing, indicating the significance of the serine residue for autocatalytic cleavage (Choi et al, 1992).…”

Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

“…With some exceptions, these compounds bind in a position favourable for nucleophilic attack by the hydroxyl group of the catalytic residue Ser B" , assisted by its αamino group, a relatively unusual catalytic arrangement [5]. Evidence for nucleophilic catalysis has come from site-directed chemical modification [6,7] and the kinetics of accumulation of an acyl-enzyme intermediate with non-specific 4-nitrophenyl ester substrates [8,9]. Some analogues of phenylacetic acid bind in an alternative position that is apparently less favourable for catalysis and the phenylacetyl group of penicillin G binds in a similar position in the inactive Asn B#%" Ala mutant enzyme [2,10].…”