Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity.
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 beta-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.
Slade
A
A
Department of Biochemistry and Genetics, School of Biomedical and Biomolecular Sciences, University of Newcastle upon Tyne, England.
Horrocks
A J
AJ
Lindsay
C D
CD
Dunbar
B
B
Virden
R
R
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010397:Penicillanic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D010399:Penicillin Amidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D011725:Pyridines / Q000494:pharmacology; D012694:Serine; D013462:Sulfur Radioisotopes
D011725:Pyridines; D013462:Sulfur Radioisotopes; D012694:Serine; D010664:Phenylmethylsulfonyl Fluoride; D010397:Penicillanic Acid; D010399:Penicillin Amidase; C029351:4-vinylpyridine; D003545:Cysteine; C004408:aminopenicillanic acid