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Archives of biochemistry and biophysics

Study of the mechanism of MF1 ATPase inhibition by fluorosulfonylbenzoyl inosine, quinacrine mustard, and efrapeptin using intermediate 18O exchange as a probe.


PMID 8424665

Abstract

The mitochondrial F1-ATPase (MF1) is known to be largely or totally inhibited by combination or reaction with one fluorosulfonylbenzoyl inosine (FSBI), quinacrine mustard, or efrapeptin per enzyme. Measurements were made with 18O in attempt to ascertain if the weak catalytic activity remaining after exposure to excess of these reagents was due to retention of some activity by the enzyme modified by these inhibitors. Any such activity could have different characteristics that might be revealed by the distribution of [18O]Pi isotopomers formed from [gamma-18O]ATP. The MF1 inhibited by FSBI showed progressive appearance of two new catalytic pathways as inhibition proceeded. Both pathways appeared to be operative in the enzyme after one beta subunit per enzyme had been modified by FSBI. A high exchange pathway showed no detectable change as ATP concentration was lowered. The lower exchange pathway showed an increase in the amount of exchange with lowering of the ATP concentration, similar to the cooperative behavior observed with the unmodified enzyme. With excess ATP more product was formed by the low exchange pathway, showing that compulsory alternation between two catalytic sites was not retained. The behavior can be explained by the ability of the modified beta subunit to undergo binding changes similar to those occurring in catalysis, with the other two beta subunits catalyzing sluggish hydrolysis by different pathways because of the asymmetry introduced by the modification. Inhibition by quinacrine mustard also resulted in the appearance of two new pathways, somewhat similar to those from FSBI inhibition. In contrast, activity remaining with excess efrapeptin present showed only one pathway like that of the native enzyme. This can be attributed to a low equilibrium concentration of free enzyme and total inhibition of MF1 combined with efrapeptin.

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