Abstract
1. Purified and reconstituted nicotinamide nucleotide transhydrogenase from beef heart mitochondria was investigated with respect to kinetic and regulatory properties in uncoupled and coupled liposomes. 2. Double reciprocal plots of initial velocities for the reduction of NAD+ by NADPH versus substrate concentrations were convergent and intersecting on or close to the abscissa, indicating a ternary complex mechanism. The effect of site-specific inhibitors indicates that the order of addition of the substrates to the enzyme is random. 3. Reconstituted transhydrogenase uncoupled by FCCP reveals kinetic properties that are indicative of energization, i.e. an increased and decreased affinity for NADP+ and NAD+, respectively, suggesting that reconstituted transhydrogenase is maintained in an activated conformation. An increased extent of coupling causes a progressively increasing change in the same direction. These results suggest that the uncoupler-dependent enhancement of the rate of reduction of NAD+ by NADPH is due to a decreased Km for NAD+. 4. Reconstituted transhydrogenase catalyzes a transhydrogenation between NADH and 3-acetylpyridine adenine dinucleotide (oxidized) in the presence of NADPH. Reconstituted transhydrogenase also also catalyzes the reduction of thio-NADP+ by NADPH in the presence of NADH. Both reactions are concluded to occur indirectly through the generation of NADP+ and NAD+, respectively, and not directly through a reduced enzyme intermediate. 5. A proton pump mechanism is proposed for transhydrogenase which involves a dimeric form of the enzyme where the two subunits are alternating in proton pumping.
Highlights
Kirsten Enander andJan RydstromS From the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden
Purified and reconstituted nicotinamide nucleotide transhydrogenase from beef heart mitochondria was investigated with respect tokinetic and regulatory properties in uncoupled and coupled liposomes
Reconstituted transhydrogenase uncoupled by FCCP reveals kinetic properties that are indicative of energization, i e . an increased and decreased affinity for NADP’ and NAD+, respectively, suggesting that reconstitutedtranshydrogenaseis maintained in an activated conformation
Summary
Vol 257, No 24, Issue of December 25, pp. 14760-14766, 1982 Printed in U.S.A. KINETICS AND REGULATION OF PURIFIED AND RECONSTITUTED TRANSHYDROGENASE FROM BEEF HEART MITOCHONDRIA*. Reconstituted transhydrogenasealsocatalyzes the reduction of thio-NADP+ by NADPH in the presence ofNADH Both reactions are concluded to occur indirectly through thegeneration of NADP’ and NAD+,respectively, and not directly through a reduced enzyme intermediate. Transfer of hydrogen between NADH and NADP+, catalyzed by mitochondrial nicotinamide transhydrogenasei,s reversible and influenced by energy generated by oxidation of respiratory substrates or ATP hydrolysis. Both directions of thereactionare affected by energization, Le. therate of reduction of NADP+ by NADH is stimulated about 10-fold and the apparenetquilibrium is increased from unityto about 500, whereas the rate of reduction of NADf by NADPH is inhibited Both directions of thereactionare affected by energization, Le. therate of reduction of NADP+ by NADH is stimulated about 10-fold and the apparenetquilibrium is increased from unityto about 500, whereas the rate of reduction of NADf by NADPH is inhibited (for review see Refs. 1and 2)
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