Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105: Effect on conformation and catalytic activity

Andrew Slade, A. Janet Horrocks, Christopher D. Lindsay, Bryan Dunbar, Richard Virden

Research output: Contribution to journalArticle

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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.
Original languageEnglish
Pages (from-to)75-80
Number of pages6
JournalFEBS Journal
Volume197
Issue number1
DOIs
Publication statusPublished - 10 Apr 1991
Externally publishedYes

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Penicillin Amidase
Sulfhydryl Compounds
Serine
Escherichia coli
Cysteine
Conformations
Catalyst activity
Enzymes
amidase
Carbocysteine
Iodoacetic Acid
Nucleophiles
Biochemistry
Proteins
Radioactivity
Chymotrypsin
Catalysis
Fluorides
Acetic Acid
Gold

Cite this

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title = "Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105: Effect on conformation and catalytic activity",
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.",
author = "Andrew Slade and Horrocks, {A. Janet} and Lindsay, {Christopher D.} and Bryan Dunbar and Richard Virden",
year = "1991",
month = "4",
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doi = "10.1111/j.1432-1033.1991.tb15884.x",
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Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105 : Effect on conformation and catalytic activity. / Slade, Andrew; Horrocks, A. Janet; Lindsay, Christopher D.; Dunbar, Bryan; Virden, Richard.

In: FEBS Journal, Vol. 197, No. 1, 10.04.1991, p. 75-80.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

T2 - Effect on conformation and catalytic activity

AU - Slade, Andrew

AU - Horrocks, A. Janet

AU - Lindsay, Christopher D.

AU - Dunbar, Bryan

AU - Virden, Richard

PY - 1991/4/10

Y1 - 1991/4/10

N2 - 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.

AB - 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.

U2 - 10.1111/j.1432-1033.1991.tb15884.x

DO - 10.1111/j.1432-1033.1991.tb15884.x

M3 - Article

C2 - 1849824

VL - 197

SP - 75

EP - 80

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 1

ER -