Energy-linked transhydrogenase. Characterization of a nucleotide-binding sequence in nicotinamide nucleotide transhydrogenase from beef heart.
Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5'-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label. NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD+ and NADP+ were inhibitory by themselves under these conditions, and the substrate analogs 5'-AMP and 2'-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2-3H]-adenosine 5'-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001-1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close to the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5'-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001-1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.
Hu
P S
PS
Department of Pharmacology, Karolinska Institute, Stockholm, Sweden.
Persson
B
B
Höög
J O
JO
Jörnvall
H
H
Hartog
A F
AF
Berden
J A
JA
Holmberg
E
E
Rydström
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D009243:NAD; C017042:8-azidoadenosine-3',5'-monophosphate; D009249:NADP; D000242:Cyclic AMP; D009250:NADP Transhydrogenases; D014357:Trypsin