Properties of bound inorganic phosphate on bovine mitochondrial F1F0-ATP synthase.
Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi <--> ATP exchange reaction with bound Pi. The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi <--> ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers.
Beharry
S
S
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Bragg
P D
PD
D000227:Adenine Nucleotides / Q000032:analysis; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D020134:Catalytic Domain; D002417:Cattle; D004121:Dimethyl Sulfoxide; D011756:Diphosphates / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010710:Phosphates / Q000737:chemistry / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000227:Adenine Nucleotides; D002021:Buffers; D011756:Diphosphates; D010710:Phosphates; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D004121:Dimethyl Sulfoxide