Synthesis and Photoaffinity Labeling of 3'(2')-O-(p-azidobenzoyl) ATP

  • Shin, Seung-Jin (Center for Molecular Catalysis, Department of Chemistry, Seoul National University) ;
  • Lee, Woo-Kyoung (Center for Molecular Catalysis, Department of Chemistry, Seoul National University) ;
  • Park, Jong-Sang (Center for Molecular Catalysis, Department of Chemistry, Seoul National University)
  • 투고 : 1997.03.15
  • 발행 : 1997.05.31

초록

A photoactive analog of ATP, 3'(2')-O-(p-azidobenzoyl)-adenosine 5-triphosphate (AB-ATP) was synthesized by chemically coupling N-hydroxysuccinimidyl-4-azidobenzoate (NHS-AB) and ATP. The utility of AB-ATP as an effective active-site-directed photoprobe was demonstrated using catalytic subunit of protein kinase A as a model enzyme. Photoincorporation of AB-ATP was saturated with apparent dissociation constant of $30{\mu}m$ and protected completely by $100{\mu}m$ of ATP. When the enzyme was covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 60% inhibition of enzyme activity was observed. These results demonstrate that AB-ATP has potential application as a probe to characterize ATP-binding proteins including protein kinases.