Section 9
Chapter 8,271

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

Muneyuki, E.; Hisabori, T.; Allison, W.S.; Jault, J.M.; Sasayama, T.; Yoshida, M.

Biochimica et Biophysica Acta 1188(1-2): 108-116


ISSN/ISBN: 0006-3002
PMID: 7947899
DOI: 10.1016/0005-2728(94)90028-0
Accession: 008270056

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) with bovine mitochondrial F1-ATPase (MF1) was examined under substoichiometric and stoichiometric conditions to investigate the relationship between the amount of bound TNP-AT(D)P and extent of inhibition on steady state ATP hydrolysis. The hydrolysis of bound TNP-ATP under substoichiometric condition proceeded slowly, with a first order rate constant of 0.014 s-1. However, hydrolysis was greatly accelerated by addition of excess ATP. The hydrolyzed product, TNP-ADP, did not dissociate from the enzyme even after the addition of excess ATP. These properties were the same for both native and nucleotide depleted enzyme. The difference spectrum induced by binding TNP-ATP to MF1 had a distinct peak 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 thermophilic F1-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 MF1 molecule. The first site, where substoichiometric 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 microM ATP was linearly suppressed as pre-loaded TNP-ATP increased. The binding of 2 mol of TNP-ATP per mol of MF1 was required to abolish ATPase activity. A model which assumes mutually-activating two catalytic sites is presented to explain these results.

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