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Catalytic site cooperativity of beef heart mitochondrial f 1 atpase correlations of initial velocity bound intermediate and oxygen exchange measurements with an alternating 3 site model


Catalytic site cooperativity of beef heart mitochondrial f 1 atpase correlations of initial velocity bound intermediate and oxygen exchange measurements with an alternating 3 site model



Journal of Biological Chemistry 257(20): 12030-12038



ISSN/ISBN: 0021-9258

Previous studies with beef heart mitochondrial F1 ATPase suggest a mechanism in which binding of substrate (ATP) at one catalytic site accelerates the release of products (ADP + Pi) from a separate catalytic site. Initial velocity studies reported here, in harmony with this mechanism, show a biphasic rate response with apparent Km values of 250 and 1.7 .mu.M and Vmax values of 54 and 2.2 .mu.mol/min per mg. Further, the suggested mechanism leads to an important and previously untested diagnostic prediction for F1 ATPase, namely that high levels of enzyme-bound product will be present during turnover at substrate concentrations well below those which give half-maximal velocity. This prediction was verified by the demonstration that most F1 ATPase molecules retain product ADP at a catalytic site when hydrolyzing ATP at concentrations less than 1/200 of that for half-maximal velocity. The mechanism also predicts a negative cooperativity of substrate binding, with a slow rate of product formation until all catalytic sites are filled. The predicted substrate binding pattern is substantiated by the demonstration reported herein that 1 catalytic site/F1 ATPase becomes saturated with only about 1 .mu.M ATP present. This singly occupied site shows slow catalytic turnover, and most of the oxygens of Pi formed at 1 .mu.M ATP have been exchanged with water. Pronounced decrease in this oxygen exchange occurs when the ATP concentration is increased to 10-20 .mu.M, which is still much lower than the concentration of ATP required for half-maximal velocity. These results cannot be satisfactorily correlated by either independent-site mechanisms or by interacting 2-site mechanisms. A simple interacting 3-site mechanism is presented that satisfactorily correlates all 3 sets of results.

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Accession: 004887256

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Related references

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