Transhydrogenase reaction

Glutaredoxin is another small ubiquitous protein with a different dithiol-active center which catalyzes GSH-disulfide transhydrogenase reactions. It is GSH-specific and cannot be reduced by thioredoxin reductase. It uses GSH and an NADPH-coupled glutaredoxin reductase to catalyze the reduction of a variety of disulfide substrates, including 2-hydroxyethyl-disulfide and ribonucleotide reductase [281]. Since GSSG inhibits the latter reaction, a high ratio of GSH to GSSG will promote the synthesis of deoxyribonucleotides, which is a likely control mechanism of DNA synthesis. 

Boger m) showed that the transhydrogenase activity catalyzed by ferredoxin-NADP reductase obtained from Bumilleriopsis filiformis, which is very similar to the spinach enzyme, is regulated by ferredoxin and that one common nicotinamide nucleotide binding site is involved in both the diaphorase and the transhydrogenase reactions. 

Fio. 1. Relationship of the energy-linked transhydrogenase reaction to oxidative phosphorylation. From Ernsteret al. 166). 

Since the equilibrium constant of the non-energy-linked transhydrogenase reaction is 0.79 (SO) and that of ATP hydrolysis is about 10 M... 

Cleland (160), steady-state kinetics of a Theorell-Chance mechanism can generally apply also to a rapid-equilibrium random mechanism with two dead-end complexes. However, in view of the data obtained with site-specific inhibitors this latter mechanism is unlikely in the case of the transhydrogenase (70, 71). The proposed mechanism is also consistent with the observation of Fisher and Kaplan (118) that the breakage of the C-H bonds of the reduced nicotinamide nucleotides is not a rate-limiting step in the mitochondrial transhydrogenase reaction. 

At neutral pH, the maximal initial velocities of the two directions of the non-energy-linked transhydrogenase reaction differ by a factor of about five, the reduction of NADP being the slower reaction (SO, 69, 68, 71, 127). Reduction of NADP by NADH is maximally active at about pH 5.5., whereas reduction of NAD by NADPH shows a pH optimum at about 7.0 (SO, 67, 71, 72 see also S2). When the reduction of NADP by NADH approaches equilibrium, the rate constant of the reaction is increased (67), indicating an activation of the transhydrogenase that is related to the accumulation of the products NAD and NADPH. It has been proposed (67, 69, 71) that this activation may involve a conversion of the enzyme from an inactive to an active conformational state, similar to that proposed to occur upon energization (see below). 

Michaelis constants of the non-energy-linked beef heart transhydrogenase reaction are 9 ftM for NADH, 40 nM for NADP , 28 )tM for NAD , and 20 pM for NADPH (68) these values are similar to those found with other AB-specific transhydrogenases (66, 89 see also 8). Dissociation constants for the E-NADH and E-NAD complexes, derived from... 

A different factor was isolated from Rhodopseudomonas spheroides by Orlando (114) which only stimulated the light-driven transhydrogenase reaction and could be replaced by thiols (116). 

Until 1973, it was generally agreed that the mitochondrial respiratory chain is incapable of oxidizing NADPH directly (103-105). NADPH oxidation was considered to occur only through the transhydrogenase reaction and with the obligatory intermediation of NAD (103-105). In 1973,... 

Fig. 15. pH dependence of NADPH oxidase, NADPH to 3-acetylpyridine adenine dinucleotide (AP-DPN) transhydrogenase, and NADH oxidase activities of submito-chondrial particles (ETP). Conditions oxidase activities were measured in the presence of 2 mM NADH or NADPH, 0.25 M sucrose, 100 mM sodium phosphate for pH values 6-0, and 100 mM sodium acetate for pH values 5.0 and 5.5. ETP concentration was 2.16 mg/ml for the NADPH oxidase, and 0.216 mg/ml for the NADH oxidase assays. The transhydrogenase reaction was measured by the Aminco-Chance spectrophotometer at 400 minus 450 nm. The extinction coefficient used for reduced 3-acetylpyridine adenine dinucleotide at 400 nm was 2300 liters mole" cm" . Media were the same as in the oxidase assays. Dotted lines indicate uncertainty about the pH 5 rates because of possible acidity damage to ETP. The ordinate refers to nanomoles of NADPH or NADH oxidized min" x mg" of ETP protein at 30°. From Hatefi and Hanstein (,S0). 

The energy expenditure of the mitochondrial ATP-driven transhydrogenase reaction has been estimated to one ATP per NADPH formed [29,30,46,53,60-62]. Since the equilibrium constant of the nonenergy-linked transhydrogenase reaction is 0.79 [2] and that of ATP hydrolysis is about 10 M [63], the equilibrium constant of the overall reaction is also of the order of 10 M. In spite of the very unfavourable equilibrium a reversibility of the energy-Unked transhydrogenase reaction has been demonstrated using ATP production [64] or uptake of lipophilic anions [65-67] as assay. 

Michelis constants of the nonenergy-linked beef heart transhydrogenase reaction are 9 /aM for NADH, 40 jaM for NADP, 28 jaM for NAD and 20 jaM for NADPH [75]. Energy-linked changes in the Michaelis constants are particularly apparent with the oxidized nicotinamide nucleotides, giving values of 6.5 and 43.5... 

Energy-linked affinity changes also seem to be of importance for the effect of certain inhibitors of transhydrogenase, e.g., metal ions [45,80]. The energy-linked transhydrogenase reaction catalyzed by submitochondrial particles is known to be inhibited by Mg " to a lesser extent than the nonenergy-linked reaction [45]. In addition, the effect of is pH-dependent with an increasing effect of the... 

Early proposals concerning the mechanism of the energy-linked transhydrogenase reaction were based on the chemical hypothesis of oxidative phosphorylation [82] and visualized the involvement of high-energy intermediates of the type 1 X, NADH I, NADP I, etc. [29,46]. These proposals, however, just as the chemical hypothesis as a whole, had to be abandoned because of lack of experimental evidence. 

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