TOPOLOGICAL ANALYSIS OF THE PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA-COLI USING PROTEOLYTIC-ENZYMES

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha-2-beta-2 structure (alpha: M(r), 54 000; beta: M(r), 48 700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha-subunit and the N-terminal 20-25 kDa region of the beta-subunit are composed of transmembranous alpha-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 alpha-subunit. The beta-subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta-subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta-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 beta-subunit. Digestion of the beta-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 alpha and beta-subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha-subunit and the C-terminal 30 kDa region of the beta-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.