A 19F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli

Abstract

d‐Lactate dehydrogenase (d‐LDH) is a membrane‐associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65, 000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of d‐LDH and its interaction with phospholipids. We incorporated 5‐fluorotryptophan (5F‐Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide‐directed mutagenesis, and studied the 5F‐Trp‐labeled enzymes using 19F‐NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin‐labeled fatty acid, which broadens the resonance from any residue within 15 Å, we have established that the membrane‐binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F‐NMR spectroscopy of wild‐type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin‐labeled fatty acid. These experiments indicate that 9–10 Phe and 3–4 Tyr residues are located near the lipid phase. Copyright © 1993 The Protein Society