Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity
| Autor: | Richard Virden, Christopher D. Lindsay, Andrew Slade, A. Janet Horrocks, Bryan Dunbar |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Iodoacetic acid
Protein Conformation Pyridines Stereochemistry Molecular Sequence Data Penicillanic Acid Sulfur Radioisotopes Biochemistry Residue (chemistry) chemistry.chemical_compound Acetic acid Escherichia coli Serine Amino Acid Sequence Cysteine chemistry.chemical_classification Chymotrypsin biology Chemistry Circular Dichroism Chemical modification Hydrogen-Ion Concentration Phenylmethylsulfonyl Fluoride Kinetics Enzyme biology.protein Acid hydrolysis Penicillin Amidase |
| Zdroj: | European Journal of Biochemistry. 197:75-80 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1991.tb15884.x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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