ATP binding in peptide synthetases: determination of contact sites of the adenine moiety by photoaffinity labeling of tyrocidine synthetase 1 with 2-azidoadenosine triphosphate.
Characterization of the nucleotide binding domain in peptide synthetases was approached by photoaffinity labeling of tyrocidine synthetase 1 (TY1) with 2-azidoadenosine triphosphate (2-azido-ATP). Exposure of TY1 in the presence of photolabel to irradiation with ultraviolet light resulted in a time-dependent covalent modification of the enzyme with a concomitant loss of catalytic activity. Inactivation was not observed if incubation was performed in the absence of either light or the nucleotide analogue. Specificity of labeling was indicated by the ability of 2-azido-ATP to serve as a substrate in the amino acid activation reaction. The modified protein was subjected to tryptic digestion, and the fragments labeled by the nucleotide analogue were purified by reverse-phase high-performance liquid chromatography. Sequence analysis identified three tryptic peptides corresponding to residues G373-K384, W405-R416, and L483-K494, derived from the N-terminal half of the TY1 sequence. As this region shows similarity to strongly conserved regions in other peptide synthetases and acyl-CoA synthetases, it is considered to be the region catalyzing aminoacyl adenylate formation. The identified sequences appear to define components of the nucleotide binding domain found in close proximity to the adenine ring in ATP. Conservation of primary structure and homology to other carboxyl-activating enzymes of this superfamily, including peptide synthetases, insect luciferases, and acyl-CoA synthetases, is discussed.
Pavela-Vrancic
M
M
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, FRG.
Pfeifer
E
E
van Liempt
H
H
Schäfer
H J
HJ
von Döhren
H
H
Kleinkauf
H
H
D000225:Adenine / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D010453:Peptide Synthases; C035180:tyrocidine synthetase; D000225:Adenine