Conformational changes of the Neurospora plasma membrane H+ATPase during its catalytic cycle.

Abstract

Evidence is presented for the existence of at least 3 different conformational states of the Neurospora plasma membrane H'ATPase during its catalytic cycle.Incubation of isolated Neurospora plasma membrane vesicles with trypsin in the absence or presence of several different ATPase reaction cycle participants or analogues thereof gives rise to different ATPase degradation patterns depending upon the ligands employed, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses.In the absence of any ligand, the ATPase (Mr -105,000) is rapidly degraded by trypsin to small, undefined fragments with M, 5 10,000.In the presence of Mg' and vanadate, a presumed transition state analogue of the aspartylphosphoryl-enzyme hydrolysis reaction, trypsin rapidly degrades the ATPase to M, -95,000 with further degradation to an M, -88,000 form occurring much more slowly, suggesting that during the enzyme dephosphorylation reaction, the enzyme is in a conformational state significantly different than it is in when unliganded.In the presence of MgADP, a nonhydrolyzable competitive inhibitor of the ATPase, the enzyme is rapidly degraded to an M, -88,000 form which is resistant to further degradation, suggesting that substrate binding is also associated with a conformational change.Finally, in the presence of Mga' plus another competitive, nonhydrolyzable ATP analogue, P,y-methylene ATP, the ATPase is rapidly degraded to an M , -95,000 form similar to that which is produced in the presence of M8' plus vanadate, and is then subsequently degraded to an M, -88,000 form similar to that produced in the presence of MgADP.The difference between the responses seen in the presence of MgADP and P,y-methylene ATP is suggested to lie in the fact that, unlike ADP, B,y-methylene ATP can participate in the formation of the transition state of the enzyme phosphorylation reaction even though it cannot phosphorylate the enzyme.On the basis of these findings, an outline of the sequence of events that occur during the catalytic cycle of the ATPase is presented.A substantial body of evidence has now accumulated which indicates that the plasma membrane of the eukaryotic microorganism Neurospora crassa contains an electrogenic.proton-