Amino acid sequence of the NAD (H)--binding region of the mitochondrial nicotinamide nucleotide transhydrogenase modified by N,N'-dicyclohexylcarbodiimide.
Purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and NAD(H) protects the enzyme against this inhibition [Phelps, D.C., and Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The tryptic digest of TH treated with [14C]DCCD showed a single radioactive peak upon FPLC chromatography. This radioactive peak was absent from tryptic digests of TH treated with [14C]DCCD in the presence of NADH. Sequence analysis of the radioactive peak showed that it contained two peptides, one derived from the other as a result of incomplete cleavage by trypsin of a lysyl-glutamyl bond. After further digestion with Staphylococcus V8 protease, the smaller radioactive fragment was isolated and sequenced. The amino acid sequence of this fragment, as determined by manual Edman degradation, was Ala-Glu-Met-Lys. The second residue was modified. Amino acid analysis and sequence studies on the radioactive tryptic peptide mixture indicated that the sequence around the DCCD-modified residue was Glu-Met-Ser-Lys-Glu-Phe-Ile-Glu-Ala-Glu-Met-Lys. In other studies, this sequence has been found in the amino acid sequence of TH as predicted from the corresponding cDNA. The DCCD-modified peptide is near the site of NAD(H) binding, as labeled with radioactive p-fluorosulfonylbenzoyl-5'-adenosine. Furthermore, there is a high degree of homology in this region between the amino acid sequences of the bovine heart TH and the alpha subunit of the Escherichia coli TH.
Wakabayashi
S
S
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037.
Hatefi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding
D002234:Carbodiimides; D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D009243:NAD; D004024:Dicyclohexylcarbodiimide; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases