Open AccessThe catalytic site is located on subunit I of the ATPase from Halobacterium saccharovorum
Author(s) -
BONET Maria Luisa,
SCHOBERT Brigitte
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17059.x
Subject(s) - protein subunit , nucleotide , atpase , binding site , enzyme , atp synthase gamma subunit , atp hydrolysis , biochemistry , active site , chemistry , biology , gene
Nucleotide‐binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of [α‐ 32 P]ATP. A high‐affinity site, located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross‐linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg 2+ ; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg 2+ and showed identical K d values; treatment that restored the activity of the ADP‐inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non‐catalytic nucleotide‐binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg 2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F 1 ‐ATPase which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.