Identification of peptides from the adenine binding domains of ATP and AMP in adenylate kinase: isolation of photoaffinity-labeled peptides by metal chelate chromatography.
Photoaffinity labeling with azidoadenine nucleotides was used to identify peptides from the ATP and AMP binding domains on chicken muscle adenylate kinase. Competition binding studies and enzyme assays showed that the 8-azido analogues of Ap4A and ATP modified only the MgATP2- site of adenylate kinase, whereas the 2-azido analogue of ADP modified the enzyme at both the ATP and AMP sites. The positions of the two nucleotide binding sites on the enzyme were deduced by isolating and sequencing the modified peptides. Photolabeled peptides were isolated by a new procedure that used metal chelate chromatography to affinity purify the photolabeled peptides prior to final purification by reverse-phase HPLC. The sequences of the peptides that were photolabeled with the 8-azido analogues corresponded to residues K28-L44, T153-K166, and T125-E135 of the chicken muscle enzyme. The residues that were present in both tryptic- and Staphylococcus aureus V-8 protease-generated versions of these peptides were assigned to the ATP binding domain on the basis of selective photoaffinity labeling with the 8-azidoadenine analogues. These peptides and an additional peptide corresponding to positions I110-K123 were photolabeled with 2-N3ADP. Since I110-K123 was photolabeled by 2-N3ADP but not by 8-N3Ap4A, it was assigned to the AMP binding domain.
Salvucci
M E
ME
United States Department of Agriculture, University of Kentucky, Lexington.
Chavan
A J
AJ
Haley
B E
BE
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000227:Adenine Nucleotides / Q000737:chemistry*; D000249:Adenosine Monophosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000263:Adenylate Kinase / Q000737:chemistry*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D011485:Protein Binding
D000227:Adenine Nucleotides; D000345:Affinity Labels; D001386:Azides; D010455:Peptides; D000249:Adenosine Monophosphate; C032235:8-azidoadenine; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D000225:Adenine