Site-specific introduction of an electroactive label into a non-electroactive enzyme (beta-lactamase I).
A cysteine residue was introduced close to the active site of beta-lactamase I by site-directed mutagenesis to replace tyrosine-105 and was subsequently modified with an electroactive SH-specific reagent, N-(2-ferrocene-ethyl)maleimide. The resulting modified enzyme became electroactive, showing good quasireversible electrochemistry which was characteristic of the attached ferrocene moiety while retaining its specific enzymatic activity. In the presence of a suicide substrate, 6beta-iodopenicillanic acid, the redox potential shifted +20 mV suggesting that the label was sensitive to changes in the active site of the enzyme.
Di Gleria
K
K
New Chemistry Laboratory, University of Oxford, UK.
Halliwell
C M
CM
Jacob
C
C
Hill
H A
HA
D001409:Bacillus cereus / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D001665:Binding Sites; D003545:Cysteine; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005296:Ferrous Compounds / Q000737:chemistry / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology*; D000075163:Metallocenes; D016297:Mutagenesis, Site-Directed; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010405:Penicillinase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D065093:beta-Lactamase Inhibitors
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005296:Ferrous Compounds; D008301:Maleimides; D000075163:Metallocenes; C100759:N-(2-ferrocene-ethyl)maleimide; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D065093:beta-Lactamase Inhibitors; D010397:Penicillanic Acid; C031528:6-iodopenicillanic acid; D010405:Penicillinase; D003545:Cysteine; C004998:ferrocene