Truong H T, Pratt E A, Ho C
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Biochemistry. 1991 Apr 23;30(16):3893-8. doi: 10.1021/bi00230a013.
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.
对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进行了研究。蛋白水解消化研究表明，与磷脂酰甘油相互作用时，D - 乳酸脱氢酶比与磷脂酰胆碱囊泡相互作用时更能在较大程度上免受胰蛋白酶消化。野生型D - 乳酸脱氢酶以及通过位点特异性寡核苷酸定向诱变在选定区域用额外色氨酸替代的突变体已用5 - 氟色氨酸进行标记。对这些标记酶与小单层磷脂囊泡相互作用的19F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可接触外部水相。通过向含有自旋标记脂肪酸作为电子受体的磷脂囊泡中添加酶和底物，研究了磷脂囊泡中酶活性的重建。自旋标记脂肪酸的多羟基硬脂酰基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，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，可以创建一个人工电子传递系统。
