Interaction of the membrane-bound D-lactate dehydrogenase of Escherichia coli with phospholipid vesicles and reconstitution of activity using a spin-labeled fatty acid as an electron acceptor: a magnetic resonance and biochemical study.
The interaction with phospholipid vesicles of the membrane-bound respiratory enzyme D-lactate dehydrogenase of Escherichia coli has been studied. Proteolytic digestion studies show that D-lactate dehydrogenase is protected from trypsin digestion to a larger extent when it interacts with phosphatidylglycerol than with phosphatidylcholine vesicles. Wild-type D-lactate dehydrogenase and mutants in which an additional tryptophan is substituted in selected areas by site-specific oligonucleotide-directed mutagenesis have been labeled with 5-fluorotryptophan. 19F nuclear magnetic resonance studies of the interaction of these labeled enzymes with small unilamellar phospholipid vesicles show that Trp 243, 340, and 361 are exposed to the lipid phase, while Trp 384, 407, and 567 are accessible to the external aqueous phase. Reconstitution of enzymatic activity in phospholipid vesicles has been studied by adding enzyme and substrate to phospholipid vesicles containing a spin-labeled fatty acid as an electron acceptor. The reduction of the doxyl group of the spin-labeled fatty acid has been monitored indirectly by nuclear magnetic resonance and directly by electron paramagnetic resonance. These results indicate that an artificial electron-transfer system can be created by mixing D-lactate dehydrogenase and D-lactate together with phospholipid vesicles containing spin-labeled fatty acids.
Truong
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HT
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Pratt
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EA
Ho
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D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003497:Cyclic N-Oxides; D004134:Dimyristoylphosphatidylcholine / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010446:Peptide Fragments / Q000302:isolation & purification; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols / Q000494:pharmacology; D011487:Protein Conformation; D013113:Spin Labels; D014357:Trypsin
D003497:Cyclic N-Oxides; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D013113:Spin Labels; C068635:1-myristoyl-2-(11-(4,4-dimethyl-3-oxy-2-ethyl-2-oxazolidinyl)undecanoyl)-sn-glycero-3-phosphocholine; C002773:dimyristoylphosphatidylglycerol; D007770:L-Lactate Dehydrogenase; D014357:Trypsin; D004134:Dimyristoylphosphatidylcholine