A structural change in the Neurospora plasma membrane [H+]ATPase induced by N-ethylmaleimide.
The reaction of N-ethylmaleimide (NEM) with Cys-532 of the Neurospora plasma membrane [H+]ATPase results in inhibition of ATP hydrolysis which is protected by MgADP (Pardo, J. P., and Slayman, C. W. (1989) J. Biol. Chem. 264, 9373-9379). To examine the conformational state of the ATPase upon NEM modification, we have used limited trypsinolysis and domain-specific antibodies. The NEM-reacted ATPase shows increased sensitivity to trypsin, particularly in the central hydrophilic region of the polypeptide thought to contain the ATP binding and phosphorylation sites. In addition, competitive enzyme-linked immunosorbent assays indicate that the C-terminal domain of the ATPase becomes more accessible to antibody binding while the N-terminal region becomes more protected. The NEM-induced structural change is accompanied by loss of the ability to form a phosphoenzyme intermediate. The change in tertiary conformation occurs specifically upon NEM reaction with Cys-532 since neither NEM modification of Cys-545 nor fluorescein 5'-isothiocyanate modification of Lys-474 alters the tryptic digestion pattern of the ATPase. Furthermore, modification of Cys-532 with the less bulky sulfhydryl reagent methyl methanethiosulfonate does not result in a detectable structural change or loss of enzymatic activity. Thus, the introduction of a relatively bulky maleimide group at Cys-532 has specific and far-reaching effects upon the structure and function of the ATPase.
Chang
A
A
Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Slayman
C W
CW
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013861:Thiocyanates; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide