Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA.
Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.
Rajasekharan
R
R
Plant Genetic Engineering Laboratory, New Mexico State University, Las Cruces 88003.
Marians
R C
RC
Shockey
J M
JM
Kemp
J D
JD
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D042962:Acyl-CoA Oxidase; D000345:Affinity Labels; D000818:Animals; D001173:Arthrobacter / Q000201:enzymology; D001386:Azides; D001426:Bacterial Proteins / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000196:embryology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism; D010777:Photochemistry; D013552:Swine
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase; D017853:Phosphotransferases (Alcohol Group Acceptor); C020021:dephospho-CoA kinase