Phospholipid requirement and pH optimum for the in vitro enzymatic activity of the E. coli P-type ATPase ZntA.
Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system.
Zimmer
Jochen
J
University of Oxford, Department of Biochemistry, Laboratory of Molecular Biophysics, South Parks Road, OX1 3QU Oxford, UK.
Doyle
Declan A
DA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D020404:Glycerophospholipids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D020404:Glycerophospholipids; D008565:Membrane Proteins; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase