Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 75-80 |
| Number of pages | 6 |
| Journal | FEBS Journal |
| Volume | 197 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 10 Apr 1991 |
| Externally published | Yes |
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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 journal › Article
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 -