Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis.
Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.
Dyson Perrins Laboratory, University of Oxford, U.K.
J E
JE
Baldwin
J B
JB
Coates
J B
JB
Halpern
M G
MG
Moloney
A J
AJ
Pratt
D000164:Acremonium / Q000201:enzymology; D000345:Affinity Labels; D001388:Aziridines; D004798:Enzymes; D007834:Lasers; D010088:Oxidoreductases; D010782:Photolysis
D000345:Affinity Labels; D001388:Aziridines; D004798:Enzymes; C060765:2-(3-(3-trifluoromethyl-3H-diazirin-3-yl)phenoxy)acetyl-S-methyloxycarbonylsulfenylcysteinyl-valine; D010088:Oxidoreductases; C038370:isopenicillin N synthetase