The dehydrogenases

The dehydrogenase catalysing the glucose-6-phosphate to 6-phosphogluconate step is specific for the phosphorylated derivative of gluconate. 

Another quite general approach is to employ a coupled assay (Figure 7-10). Typically, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled. 

These dehydrogenases use nicotinamide adenine dinucleotide (NAD ) or nicotinamide adenine dinucleotide phosphate (NADP )—or both—and are formed in the body from the vitamin niacin (Chapter 45). The coenzymes are reduced by the specific substrate of the dehydrogenase and reoxidized by a suitable electron acceptor (Figure 11-4). They may freely and reversibly dissociate from their respective apoenzymes. 

As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADHj are produced for each molecule of acetyl-CoA catabohzed in one mrn of the cycle. These reducing equivalents are transferred to the respiratory chain (Figure 16-2), where reoxidation of each NADH results in formation of 3 ATP and reoxidation of FADHj in formation of 2 ATP. In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase. 

Inhibitors should not be present in the reagent solution. Examples are the dehydrogenase inhib-... 

Conversion of methanol into formaldehyde by methanol dehydrogenase. A complex array of genes is involved in this oxidation and the dehydrogenase contains pyrroloquinoline quinone (PQQ) as a cofactor (references in Ramamoorthi and Lidstrom 1995). Details of its function must, however, differ from that of methylamine dehydrogenase that also contains a quinoprotein—tryptophan tryptophylquinone (TTQ). 

Pereira MM, Carita JN, Teixeira M. 1999. Membrane-bound electron transfer chain of the ther-mohalophilic bacterium Rhodothermus marinus Characterization of the iron- sulfur centers from the dehydrogenases and investigation of the high-potential iron- sulfur protein function by in vitro reconstitution of the respiratory chain. Biochemistry 38 1276. 

While this anode is not useful in the context of implantable fuel cells, it is of interest because methanol is an attractive anodic fuel due to its availability and ease of transport and storage. The oxidation of one equivalent of methanol requires the reduction of three equivalents of NAD+ to NADH. As the NADH cofactor itself is not a useful redox mediator, a benzylviologen/diaphorase redox cycle, with a redox potential of 0.55 V vs SCE at pH 7, was used to regenerate NAD+ for use by the dehydrogenases, as depicted in Fig. 12.10. 

M12. McNair Scott, D. B., and Cohen, S. S., The oxidative pathway of carbohydrate metabolism in Escherichia coli. 5. Isolation and identification of ribulose-5-phosphate produced from 6-phosphogluconate by the dehydrogenases of Escherichia coli. Biochem. J. 65, 686-689 (1957). 

P450 can also catalyze hydroxylation of a carbon-hydrogen bond a to an oxygen atom in an alcohol. But, in contrast to the ethers, the primary oxidants of alcohols appear not to be the P450s but other enzymes like the dehydrogenases as will be discussed later. 

The natural substrate for the dehydrogenase, glyceraldehyde-3-phosphate (G-3-P), had been synthesized earlier by Hermann Fischer, Emil Fischer s son, and Baer in 1932. In 1934 Meyerhof and Lohmann synthesized hexose diphosphate, establishing it to be fructose 1,6 bisphosphate (F-l, 6 bis P). With F-1,6 bisP as substrate and hydrazine to trap the aldehydic and ketonic products of the reaction, G-3-P was identified in the mixture of G-3-P and dihydroxyacetone phosphate which resulted. Triose phosphate isomerase was then isolated and the importance of phosphorylated 3C derivatives established. 

F. M. Dickinson, G. W. Haywood, The Effects of Mg2+ on Certain Steps in the Mechanism of the Dehydrogenase and Esterase Reactions Catalysed by Sheep Liver Aldehyde Dehydrogenase. Support for the View That Dehydrogenase and Esterase Activities Occur at the Same Site on the Enzyme , Biochem. J. 1986, 233, 877-883. 

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