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.
Department of Chemistry, Connecticut College, 270 Mohegan Avenue, New London, CT 06320, USA. brbra@conncoll.edu
Bruce R
BR
Branchini
Martha H
MH
Murtiashaw
Jill N
JN
Carmody
Emily E
EE
Mygatt
Tara L
TL
Southworth
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