A F-19-NMR STUDY OF THE MEMBRANE-BINDING REGION OF D-LACTATE DEHYDROGENASE OF ESCHERICHIA-COLI

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory e nzyme of Escherichia coli. The protein is composed of 571 amino acid r esidues with a flavin adenine dinucleotide (FAD) cofactor, has a molec ular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the struct ure 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 tryptopha n is substituted into a specific site by oligonucleotide-directed muta genesis, and studied the 5F-Trp-labeled enzymes using F-19-NMR spectro scopy. In this way, information was obtained about the local environme nt at each native and substituted tryptophan site. Using a nitroxide s pin-labeled fatty acid, which broadens the resonance from any residue within 15 angstrom, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but i s not continuous within this region. This conclusion is strengthened b y the results of F-19-NMR spectroscopy of wild-type enzyme labeled wit h fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 34 Tyr residues are located near the lipid phase.