Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a stable and potent inhibitor of firefly luciferase.
In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5'-O-[(N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent (Ki = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.
Branchini
Bruce R
BR
Department of Chemistry, Connecticut College, 270 Mohegan Avenue, New London, CT 06320, USA. brbra@conncoll.edu
Murtiashaw
Martha H
MH
Carmody
Jill N
JN
Mygatt
Emily E
EE
Southworth
Tara L
TL
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives*; D000818:Animals; D004355:Drug Stability; D049454:Firefly Luciferin / Q000031:analogs & derivatives*; D007700:Kinetics; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors*; D018716:Molecular Mimicry; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013451:Sulfonic Acids / Q000138:chemical synthesis* / Q000494:pharmacology
D010446:Peptide Fragments; D013451:Sulfonic Acids; D000249:Adenosine Monophosphate; D049454:Firefly Luciferin; C005741:sulfamic acid; D049409:Luciferases, Firefly; D010447:Peptide Hydrolases