On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase.
Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism / Q000494:pharmacology; D000495:Allosteric Site; D000818:Animals; D002417:Cattle; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014443:Tyrosine
D000345:Affinity Labels; D046911:Macromolecular Substances; D014443:Tyrosine; D006639:Histidine; D000244:Adenosine Diphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000241:Adenosine