Inhibition of the plasma membrane [H+]-ATPase of Neurospora crassa by N-ethylmaleimide. Protection by nucleotides.

Abstract

Pre-incubation of the plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent, N-ethylmaleimide (NEM), leads to a marked inhibition of ATPase activity. Loss of activity depends upon preincubation pH and follows pseudo-first order kinetics with respect to NEM concentration. MgATP, the physiological substrate for ATPase activity, protects against NEM inactivation with an average dissociation constant of 1.5 mM at 0 degrees C. This value agrees well with the measured Km for MgATP hydrolysis (1.3 mM at 30 degrees C and 0.9 mM at 15 degrees C). MgADP also protects against NEM inhibition with an average KD of 0.18 mM at 0 degrees C; MgADP is a competitive inhibitor of enzyme activity, with a Ki of 0.08 mM at 30 degrees C and 0.09 mM at 15 degrees C. Free ATP and ADP, as well as other Mg nucleotides (MgGTP, MgCTP, and MgUTP) which are hydrolyzed at much slower rates than MgATP, exert smaller protective effects. These results suggest that nucleotides protect against NEM inhibition by binding to the catalytic site of the ATPase. NEM can therefore be used as a probe to study the nature of enzyme-ligand interactions.