Definition of a nucleotide binding site on cytochrome c by photoaffinity labeling.
We have used TNP-8N3-AMP (2'(3')-O-(2,4,6-trinitrophenyl)-8-azidoadenosine monophosphate) and TNP-8N3-ATP to probe the ATP binding site(s) of cytochrome c. Irradiation of cytochrome c with close to stoichiometric amounts of TNP-8N3-AMP at low ionic strength derivatized approximately half of the protein, with the mono-derivatized species being associated with four peaks (B, 6%; C, 17%; D, 24%; E, 4%) eluted from a cation exchange column. Irradiation in the presence of ATP suggested that the main peaks C and D resulted from more specific nucleotide binding. Thermolysin digestion and TNP-peptide purification and sequencing revealed that peak C was associated with derivatization of mainly Lys-86 and to a lesser extent Lys-72 and peak D with mainly Lys-87 and less so with Lys-72. Minor peaks B and E could not be identified. TNP-8N3-ATP photolabeling produced similar results, showing favored interaction of the adenyl ring with Lys-86 and Lys-87 and to a lesser extent with Lys-72. The results are compatible with previous findings that suggest that the principal locus of ATP binding is at nearby Arg-91 (Corthesy, B. E., and Wallace, C. J. A.(1986) Biochem. J. 236, 359-364). Molecular modeling with energy-minimized docking of ATP between the 60s helix and the 80s stretch with the gamma-phosphate constrained to interact with Arg-91, places the 8 position close to Lys-86 and Lys-87 in the anti conformation about the glycosidic bond and to Lys-72 in the syn conformation, and the ribose hydroxyls within H-bonding distance of Glu-69.
McIntosh
D B
DB
Medical Research Council Biomembrane Research Unit and Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa.
Parrish
J C
JC
Wallace
C J
CJ
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010777:Photochemistry; D011487:Protein Conformation; D013816:Thermodynamics
D000345:Affinity Labels; D001386:Azides; D003574:Cytochrome c Group; D009711:Nucleotides; C058476:O(2',3')-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate; C073484:2',3'-O-(2,4,6-trinitrophenyl)-8-azido-AMP; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate