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Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-(p-(fluorosulfonyl)benzoyl)adenosine

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00335a017· OSTI ID:6381222
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Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-(p-(fluorosulfonyl)benzoyl)adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. (/sup 3/H)FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of (3H)FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by (/sup 14/C)DCCD. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

Research Organization:
Scripps Clinic and Research Foundation, La Jolla, CA
OSTI ID:
6381222
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 14; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADENOSINE
ADP
AFFINITY
AMP
ANIMALS
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CATTLE
CELL CONSTITUENTS
COENZYMES
COVALENCE
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIGANDS
MAMMALS
MITOCHONDRIA
NAD
NADP
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANOIDS
OXIDOREDUCTASES
REACTION KINETICS
RIBOSIDES
RUMINANTS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES