Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an α2β2 structure (α: Mr, 54 000; β: Mr, 48 700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the α subunit and the N-terminal 20–25 kDa region of the β subunit are composed of transmembranous α-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the α subunit. The β subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both α and β subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The β subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the β subunit. Digestion of the β subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the α and β subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the α subunit and the C-terminal 30 kDa region of the β subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.