THE KINETICS AND MECHANISM OF BEEF HEART MITOCHONDRIAL ATPASE: CHROMIUM NUCLEOTIDES, CHEMICAL MODIFICATION AND ISOTOPE EXCHANGE STUDIES

The mechanism of beef heart mitochondrial ATPase (F(,1)) was studied using chromium (III) substituted substrate analogs. Incubation of F(,1) with monodentate CrADP and P(,i) resulted in the formation of a tightly bound (\u2732)P(,i)-CrADP-enzyme complex. The enzyme slowly released a product, P(,i)-CrADP. This product was also produced by F(,1) catalyzed partial hydrolysis of bidentate CrATP. These results indicated that nonmembrane bound F(,1) was capable of net synthesis of what may be an ATP synthesis and hydrolysis intermediate analog. The F(,1)-dependent formation of the complex was taken as evidence that the soluble ATPase can function in ATP synthesis as well as ATP hydrolysis. The mono- and bidentate forms of CrADP were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of CrATP. Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by F(,1). These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms. The rates of chromium nucleotide isomer interconversion were studied as a function of pH, ionic strength and temperature. Nucleotide isomers were separated using high voltage electrophoresis and gel filtration chromatography. The rate of conversion of monodentate CrADP to the bidentate complex increased with increasing pH, temperature and ionic strength. It was found that pH values above 7.0, the chromium complexes rapidly decomposed even at 4(DEGREES)C. Optimal stability for CrADP complexes was found to be at pH 3.5 at 4(DEGREES)C. It was found that the conversion of monodentate CrADP to bidentate CrADP required the removal of one proton by the solvent and the activation energy for the conversion was 5.5 Kcal mole(\u27-1). A possible mechanism for isomer conversion is discussed. We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F(,1)) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalize the P(,i) (DBLARR) ATP exchange reaction. The ATP hydrolysis activity of isolated F(,1) was lost upon chemical modification by phenylglyoxal, butanedione, 7-chloro-4-nitrobenzene-2-oxa-1,2-diazole, or cold inactivation. The F(,1) thus modified was able to catalyze an ADP-dependent P(,i) (DBLARR) ATP exchange reaction. However, the ability of the enzyme to produce P(,i)-CrADP from phosphate and monodentate CrADP was unimpaired by the loss of hydrolysis activity. The P(,i) (DBLARR) ATP exchange catalyzed by modified F(,1) was shown to be totally inhibited by the F(,1)-specific antibiotic efrapeptin but was not inhibited by oligomycin or the uncoupler dinitrophenol. The exchange reaction is inhibited by AMP-PNP. The P(,i) (DBLARR) ATP exchange reaction catalyzed by NBD-modified and cold-treated F(,1) was characterized in detail. The maximum amount of exchange is detected when 9% of the original hydrolysis activity remains. The pH optimum for both modified enzymes are 8.0 Both ATP and ADP exert substrate inhibition but not P(,i). The k(,m) for P(,i) is 2.0 mM. The effects of various metals, anions and amino acid-specific reagents were also studied. The implications of these data with regard to the mechanism of ATP synthesis are discussed