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: Mr, 54,000; beta: Mr, 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.
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
R C
RC
Tong
N A
NA
Glavas
P D
PD
Bragg
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin; D004926:Escherichia coli / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology; D008969:Molecular Sequence Data; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D012995:Solubility; D014357:Trypsin
D010446:Peptide Fragments; D009250:NADP Transhydrogenases; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin