Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl[3H]adenosine at 65 degrees C.
Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with [3H]FSBA by about 90%, greater than 20 mol of [3H]SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM [3H]FSBA at 65 degrees C and pH 6.4, about 10 mol of [3H]SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985) Abstracts International Symposium on Energy Transducing ATPases, Kobe, Japan, p. 84), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.
Bullough
D A
DA
Yoshida
M
M
Allison
W S
WS
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006180:Proton-Translocating ATPases / Q000032:analysis* / Q000037:antagonists & inhibitors; D013696:Temperature; D014316:Tritium; D014357:Trypsin / Q000494:pharmacology
D000345:Affinity Labels; D014316:Tritium; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D000241:Adenosine