NADH case studies

In the following year, Cleland and his coworkers reported further and more emphatic examples of the phenomenon of exaltation of the a-secondary isotope effects in enzymic hydride-transfer reactions. The cases shown in Table 1 for their studies of yeast alcohol dehydrogenase and horse-liver alcohol dehydrogenase would have been expected on traditional grounds to show kinetic isotope effects between 1.00 and 1.13 but in fact values of 1.38 and 1.50 were found. Even more impressively, the oxidation of formate by NAD was expected to exhibit an isotope effect between 1.00 and 1/1.13 = 0.89 - an inverse isotope effect because NAD" was being converted to NADH. The observed value was 1.22, normal rather than inverse. Again the model of coupled motion, with a citation to Kurz and Frieden, was invoked to interpret the findings. 

Examination of one real-life case may benefit the reader s understanding. Strittmatter studied the primary kinetic isotope effects arising in the NADH-dependent cytochrome bs reductase (EC 1.6.2.2). The oxidation of NADH and subsequent reduction of cytochrome bs is facilitated by the enzyme-bound FAD group, and the kinetics of the direct transfer of a hydrogen from the A-face (or pro-R) of NADH to the flavin can be monitored by the loss of the 340 run absorbance of the NADH s dihydropyridine ring. Using deuterated isotopic isomers of NADH and several related compounds, Strittmatter obtained the primary kinetic isotope effect data compiled in the table below. 

A very useful complement to enzyme assays as described above is histochemical study, which can provide additional information [76]. In particular, because it is possible to measure the activity cell per cell, histochemistry permits, in the case of a heteroplasmic population of mitochondria, the detection of even a small number of affected cells, which may have remained undetected by biochemical assays. Spectacular images showing, side-by-side, cells endowed with either high or absent enzyme activity can be obtained. The limitation of the method is in part due to the few activities possibly measured (essentially complex IV, succinic dehydrogenase, and less specifically, ATPase and NADH reductase) and to the fact that it is poorly quantitative. Histochemical investigations are performed under selected conditions (e.g., substrate concentrations, pH), which often differ from those used for enzyme assays, thus possibly introducing discrepancy between the two approaches. 

NADH as an end product. This implicates oxidized malic acid, either pyruvic or oxaloacetic acid, as another end product. By adding commercial preparations of L-lactic dehydrogenase or malic dehydrogenase to the reaction mixture, Morenzoni (90) concluded that the end product was pyruvic acid. Attempts were then made to show whether two enzymes—malate carboxy lyase and the classic malic enzyme, malate oxidoreductase (decarboxylating), were involved or if the two activities were on the same enzyme. The preponderance of evidence indicated that only one enzyme is involved. This evidence came from temperature inactivation studies, heavy-metal inhibition studies, and ratio measurements of the two activities of partially purified preparations of Schiitz and Radlers malo-lactic enzyme (76, 90). This is not the first case of a single enzyme having two different activities (91). 

It is known from electrochemical studies that fullerenes are easily reduced. Up to 6 electrons can be added reversibly [19], and, as mentioned earlier, the excited states are even more easily reduced. A large number of electron donors were investigated including aromatic and alkyl amines [29,43,79,119-140,152,161], ni-troxide radicals [57,117], suspensions of Ti02 [118], polyaromatic compounds, [19,127] organo-silicon compounds, [133,158] phenothiazine, [133] acridine [145,154], (3-carotene [141], tetrathiafulvalenes [146], tetraethoxyethene [147], phthalocyanines [148], porphyrines [151,153], NADH and analogues [150,154, 155], borates [156,159], and naphtoles [23] to name a few representative cases. 

The localization at the cellular and subcellular levels of the liver and kidney enzymes responsible for this reduction as well as the mechanism of action have been studied by Aymard et al. (69) the liver enzymic activity is localized mainly in the cytosol while the kidney enzymatic activity appears to be associated mainly with the cell membranes and nuclei. In both cases, the enzyme is thermolabile and its activity is enhanced by adding NADH. 

Studies on the mechanism of NADPH-cytochrome P-450 reductase have been carried out thus far only with the trypsin- or lipase-solubilized forms. Assuming that this enzyme is composed of several semi-autonomous domains, and assuming further that modification during solubilization is restricted to the domain involved in the interaction with cytochrome P-450, then, as was the case with NADH-cytochrome bs reductase, mechanism studies on the soluble enzyme will contribute to the ultimate understanding of the operation of the reconstituted system. The fact that the soluble reductase is composed of a single polypeptide chain gives hope that the modification is a subtle one. 

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