CATALYTIC COOPERATIVITY OF BEEF-HEART MITOCHONDRIAL F1-ATPASE REVEALED BY USING 2',3'-O-(2,4,6-TRINITROPHENYL)-ATP AS A SUBSTRATE - AN INDICATION OF MUTUALLY ACTIVATING CATALYTIC SITES

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) with bo vine mitochondrial F-1-ATPase (MF(1)) was examined under substoichiome tric and stoichiometric conditions to investigate the relationship bet ween the amount of bound TNP-AT(D)P and extent of inhibition on steady state ATP hydrolysis. The hydrolysis of bound TNP-ATP under substoich iometric condition proceeded slowly, with a first order rate constant of 0.014 s(-1). However, hydrolysis was greatly accelerated by additio n of excess ATP. The hydrolyzed product, TNP-ADP, did not dissociate f rom the enzyme even after the addition of excess ATP. These properties were the same for both native and nucleotide depleted enzyme. The dif ference spectrum induced by binding TNP-ATP to MF(1) had a distinct pe ak at 410 nm and a deep trough at 395 nm, which were similar to those induced when TNP-ATP bound to the isolated beta subunit of the thermop hilic F-1-ATPase. The magnitude of difference spectra as a function of TNP-ATP concentration suggested the presence of at least two types of binding sites on the MF(1) molecule. The first site, where substoichi ometric TNP-ATP was hydrolyzed, had a very high affinity for TNP-ATP. TNP-AT(D)P bound to this site did not dissociate even in the presence of excess ATP. TNP-AT(D)P bound to the second site dissociated slowly when excess ATP was added. The steady state ATPase activity at 100 mu M ATP was linearly suppressed as pre-loaded TNP-ATP increased. The bin ding of 2 mol of TNP-ATP per mol of MF(1) was required to abolish ATPa se activity. A model which assumes mutually-activating two catalytic s ites is presented to explain these results.