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.
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.
复制标题
大肠杆菌膜结合 D-乳酸脱氢酶与磷脂囊泡的相互作用以及使用自旋标记脂肪酸作为电子受体重建活性：磁共振和生化研究。
作者：
Truong,HT;Pratt,EA;Ho,C
卡内基梅隆大学生物科学系，4400 Fifth Avenue，Pittsburgh，Pennsylvania 15213接收于1990年10月15日;修订于1990年11月29日摘要：研究了大肠杆菌膜结合呼吸酶D-乳酸脱氢酶与磷脂囊泡的相互作用。蛋白水解消化研究表明，D-乳酸脱氢酶与磷脂酰甘油的相互作用比与磷脂酰胆碱囊泡的相互作用更大程度上受到胰蛋白酶消化的保护。野生型D-乳酸脱氢酶和突变体，其中一个额外的色氨酸被取代，在选定的地区，由位点特异性色氨酸定向诱变已标记5-氟色氨酸。这些标记的酶与小的单层磷脂囊泡的相互作用的19 F核磁共振研究表明，色氨酸243，340和361暴露于脂质相，而色氨酸384，407和567可接近外部水相。通过向含有自旋标记脂肪酸作为电子受体的磷脂囊泡中加入酶和底物，研究了磷脂囊泡中酶活性的重建。自旋标记脂肪酸的羟基基团的还原已通过核磁共振间接监测，并通过电子顺磁共振直接监测。这些结果表明，D-乳酸脱氢酶（d-LDH）1是一种分子量为65000的黄素酶，位于大肠杆菌细胞膜的胞质侧，通过将D-乳酸脱氢酶和D-乳酸与含有自旋标记脂肪酸的磷脂囊泡混合，可以建立人工电子传递系统。它在电子转移反应中催化D-乳酸的氧化，该反应与各种氨基酸和糖主动转运到由E. coli（巴恩斯和卡巴克，1971; Futai，1973; Kohn和卡巴克，1973）。d-LDH的活性通过多种脂质和去污剂增强（Fung等人，1979; Kovatchev等人，
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania15213 Received October 15, 1990; Revised Manuscript Received November 29, 1990 abstract: 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 trypsindigestion 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 havebeen 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 byadding 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 togetherwith phospholipid vesicles containing spin-labeled fatty acids.D-Lactate dehydrogenase (d-LDH) 1 is a flavin-containing enzyme with a molecular weight of 65 000, located on the cytoplasmic side of the Escherichia coli membrane. It cata-lyzes the oxidation of D-lactate in an electron-transfer reaction that is coupled to the active transport of various amino acids and sugars into membrane vesicles prepared from E. coli (Barnes & Kaback, 1971; Futai, 1973; Kohn & Kaback, 1973). The activity of d-LDH is enhancedby a wide variety of lipids and detergents (Fung et al., 1979; Kovatchev et al.,