Modulation by ADP and Mg2+ of the inactivation of the F1-ATPase from the thermophilic bacterium, PS3, with dicyclohexylcarbodiimide.

TitleModulation by ADP and Mg2+ of the inactivation of the F1-ATPase from the thermophilic bacterium, PS3, with dicyclohexylcarbodiimide.
Publication TypeJournal Article
Year of Publication1983
AuthorsYoshida, M, Allison, WS
JournalJ Biol Chem
Volume258
Issue23
Pagination14407-12
Date Published1983 Dec 10
ISSN0021-9258
KeywordsAdenosine Diphosphate, Bacteria, Carbodiimides, Cations, Divalent, Dicyclohexylcarbodiimide, Magnesium, Proton-Translocating ATPases
Abstract

The soluble F1-ATPase from the thermophilic bacterium PS3 (TF1) contains no endogenous adenine nucleotides and contains about 0.2 g ions of Mg2+/mol which resists removal by repeated centrifugation-elution on columns of Sephadex G-50. The isolated enzyme will not bind additional Mg2+ added in the absence of adenine nucleotides nor is the rate of inactivation of the isolated enzyme by dicyclohexylcarbodiimide (DCCD) affected by the addition of Mg2+. When ADP is added to isolated TF1, a 1:1 TF1 X ADP complex is formed which is stable to repeated gel permeation on columns of Sephadex G-50 subjected to centrifugation-elution. On formation of the 1:1 TF1 X ADP complex, the rate of inactivation of the enzyme by DCCD is accelerated 6-fold. The rate of inactivation of the 1:1 TF1 X ADP complex by DCCD is not further stimulated in the presence of 2 mM ADP which indicates that the binding of ADP to a single site in the enzyme is sufficient to promote maximal stimulation of the inactivation. Addition of Mg2+ to the 1:1 TF1 X ADP complex results in the binding of about 1 g ion of Mg2+/mol of enzyme. The 1:1:1 TF1 X ADP X Mg2+ complex thus formed is sluggishly inactivated by DCCD. When the Mg2+ is removed from the TF1 X ADP X Mg2+ complex by treatment with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, the rate of inactivation of the enzyme by DCCD is accelerated 4-fold. Other divalent metal ions protect the 1:1 TF1 X ADP complex against inactivation by DCCD. Of these, Mn2+, Zn2+, Co2+, and Cd2+, which are about as equally effective as Mg2+ as cofactors for the hydrolytic reaction when present at 0.2 mM, offer about equal protection of the complex against inactivation by DCCD also when present at 0.2 mM. These results indicate that the binding site for ADP in the 1:1 TF1 X ADP complex is a catalytic site. TF1, inactivated by 92% with DCCD, has the same capacity to bind ADP as the active enzyme, forming a tight 1:1 TF1 X ADP complex which is stable to repeated centrifugation-elution on columns of Sephadex G-50. The 1:1 TF1 X ADP complex retains its capacity to bind Mg2+ to form the 1:1:1 TF1 X ADP X Mg2+ complex after it is inactivated by 88% with DCCD.

Alternate JournalJ. Biol. Chem.
Citation Key3256
PubMed ID6227624
Grant ListGM-16974 / GM / NIGMS NIH HHS / United States