Enzyme inhibitors oligomycin

B. The ATP synthase inhibitor oligomycin binds directly to the enzyme complex and plugs up the H channel, which blocks ATP formation. 

One striking characteristic of the coupling ATPase of energy-transducing membranes, apart from the extraordinarily large number of different polypeptide subunits, is the existence of two different polypeptides involved in the response of the enzyme to the inhibitor oligomycin. One binds the inhibitor, the other, separated in space from the former by possibly as much as 10 to 15 A, confers oligomycin sensitivity to the entire enzyme complex. How could the transfer of information between these two polypeptide subunits and their concerted interaction with the ATPase proper be visualized ... 

The activity of the ATP-forming enzyme complex V is usually assessed by determining the reverse reaction ATP — ADP + Pi. The reaction is coupled to reactions catalyzed by pyruvate kinase (ADP + phosphoenolpyruvate —> pyruvate + ATP) and lactate dehydrogenase (pyruvate + NADH — lactate -F NAD+). This final reaction can be followed spectrophotometrically by measuring NADH at 340 nm. The activity of complex V (ATPase) can be derived from the rate of NADH conversion in the presence and absence of the specific complex V inhibitor oligomycin. 

The activity of complex V (ATP synthase) can be conveniently measured in the reverse direction, ATP hydrolysis with a coupled assay thereafter described [72] (Fig. 3.8.6). The use of oligomycin, a specific inhibitor of the enzyme, allows discrimination of the mitochondrial enzyme from any nonmitochondrial ATPases. 

Another class of phosphorylation inhibitors functions by blocking the ATP-synthase enzyme directly. Inhibitors of this type include the antibiotic oligomycin. If mitochondria are treated with oligomycin, ADP no longer is able to increase the rate of respiration (fig. 14.16b). Oligomycin does not block the stimulation of respiration caused by an uncoupler, demonstrating that the two types of inhibitors of phosphorylation act by different mechanisms. 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

Other inhibitors of the enzyme also had similar effects on the ac-stimu-lated activity. Ouabain (50% inhibition at 0.35 xM), oligomycin (8.0 xM), ouabagenin (3 jlM), and vanadate (20 jlM) all inhibited the ac-stimulated pumping activity. 4,4 -Bis(isothiocyano)-2,2 -distilbenesulfonate (DIDS), an inhibitor of band 3 protein-mediated Li+/Na+ exchange, had no effect, nor did phloretin at micromolar concentrations. These results show that Na, K-ATPase is responsible for the observed effects. 

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