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N-terminal sequence and distal histidine residues are responsible for pH-regulated cytoplasmic membrane binding of

Donna K Mahnke-Zizelman1, Richard L Sabina

  • 1Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

The Journal of Biological Chemistry
|September 6, 2002
PubMed

Insights

Full-sized AMP deaminase 3 (AMPD3) isoform E binds to cell membranes via histidine residues, with pH influencing this interaction. This pH-responsive binding affects enzyme activity and may regulate adenylate metabolism.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Mammalian AMP deaminase 3 (AMPD3) enzymes are known to interact with cellular membranes, affecting their conformation and function.
  • Previous studies may have used N-truncated AMPD enzymes due to proteolytic sensitivity, potentially leading to inaccurate conclusions about full-length enzyme behavior.

Purpose of the Study:

  • To characterize the reversible association of full-sized human recombinant AMPD3 isoform E with the cytoplasmic membrane.
  • To investigate the role of pH and specific amino acid residues in this membrane interaction and its effect on enzyme activity.

Main Methods:

  • Utilized erythrocyte ghosts to study the membrane association of full-sized recombinant AMPD3 isoform E.
  • Investigated the effect of neomycin on protein-lipid interaction and catalytic activity.
  • Employed diethyl pyrocarbonate (DEPC) modification to probe the role of histidine residues and pH.
  • Analyzed difference spectra to identify DEPC-modified histidine residues.
  • Compared isoform E with AMPD1 and N-truncated AMPD3 variants.

Main Results:

  • Membrane-bound AMPD3 isoform E showed reduced catalytic activity.
  • Neomycin disrupted the protein-lipid interaction and restored catalytic activity at low micromolar concentrations.
  • Membrane association of isoform E was inversely correlated with physiological pH.
  • DEPC modification abolished membrane binding, an effect reversible by hydroxylamine, indicating histidine involvement.
  • Histidine residues mediate pH-responsive binding to anionic membrane charges, potentially phosphatidylinositol 4,5-bisphosphate.
  • These properties are specific to isoform E and depend on the N-terminal 48 amino acids.

Conclusions:

  • Protonated histidine residues mediate the pH-responsive binding of AMPD3 isoform E to the cytoplasmic membrane.
  • This pH-dependent membrane partitioning is a novel mechanism for regulating adenylate catabolism at branch points.
  • The N-terminus of isoform E is crucial for this specific membrane interaction, distinguishing it from other isoforms and truncated variants.

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