Specificity, of dehydrogenases

Coenzyme Specificity of Dehydrogenases. A number of dehydrogenases have been reported to require DPN, others are specific for TPN, and several react with both, although not necessarily at the same rate. Examples have already been cited of enzymes that catalyze identical reactions but possess different coenzyme requirements. An additional means for studying pyridine nucleotide reactions became available with desamino DPN. Certain dehydrogenases (e.g., liver alcohol dehydrogenase) react at equal rates with DPN and desamino DPN. Others (as... 

The retinol that is delivered to the retinas of the eyes in this manner is accumulated by rod and cone cells. In the rods (which are the better characterized of the two cell types), retinol is oxidized by a specific retinol dehydrogenase to become 2iW-trans retinal and then converted to 11-eis retinal by reti-... 

Compartmentation of these reactions to prevent photorespiration involves the interaction of two cell types, mescrphyll cells and bundle sheath cells. The meso-phyll cells take up COg at the leaf surface, where Og is abundant, and use it to carboxylate phosphoenolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 22.30). This four-carbon dicarboxylic acid is then either reduced to malate by an NADPH-specific malate dehydrogenase or transaminated to give aspartate in the mesophyll cells. The 4-C COg carrier (malate or aspartate) then is transported to the bundle sheath cells, where it is decarboxylated to yield COg and a 3-C product. The COg is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another COg (Figure 22.30). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of COg uptake (C3 plants). 

Minteer and co-workers have also exploited the broad substrate specificity of PQQ-dependent alcohol dehydrogenase and aldehyde dehydrogenase from Gluconobacter species trapped within Nahon to oxidize either ethanol or glycerol at a fuel cell anode [Arechederra et al., 2007]. Although the alcohol dehydrogenase incorporates a series of heme electron transfer centers, it is unlikely that many enzyme molecules trapped within the mediator-free Nahon polymer are electronically engaged at the electrode. 

Weckbecker, A. and Hummel, W. (2006) Cloning, expression, and characterization of an (/ (-specific alcohol dehydrogenase from Lactobacillus kefir. Biocatalysis and Biotransformation, 24 (5), 380-389. 

Huang, Y.J. and Komuniecki, R. (1997) Cloning and characterization of a putative testis-specific pyruvate dehydrogenase beta subunit from the parasitic nematode, Ascaris suum. Molecular and Biochemical Parasitology 90, 391-394. 

Moore SA, Baker HM, Blythe TJ, Kit-son KE, Kitson TM, Baker EN. Sheep liver cytosolic aldehyde dehydrogenase the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases. Structure 1998 6 1541-1551. 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

R is an electron-donor substrate such as purine or xanthine and A is an electron acceptor such as 02 or NAD+. It is thought that the in vivo mammalian form of xanthine oxidase uses NAD+ as acceptor and is therefore, more appropriately named xanthine dehydrogenase. No evidence exists for a dehydrogenase form of aldehyde oxidase. The specificities of xanthine oxidase and aldehyde oxidase have been extensively catalogued (96), and the mechanism and properties of these enzymes have been reviewed (97, 98). 

While most alkaloids do not contain aldehydes when they enter mammalian, microbial, or plant tissues, this functional group may become important when formed as a metabolite of alcohols (via alcohol dehydrogenase) or amines (via oxidative dealkylation and oxidative deamination). Aldehyde dehydrogenases catalyze oxidation of aldehydes to the corresponding carboxylic acids. The physical properties, catalytic mechanism, and specificity of this group of enzymes has been reviewed (99). The general reaction catalyzed by aldehyde dehydrogenase is seen in Eq. (9). 

The mechanism proposed for the aldehyde dehydrogenases includes an enzyme-bound hemiacetal intermediate, possibly via a thioester bond with a cysteine (100). The specificity of the enzyme for aldehydes is quite broad. Apparent Km values for many aliphatic and aromatic aldehydes are in the micromolar range, with the highest reaction velocities observed for aldehydes with electron-with-drawing substituents on the a carbon for aliphatic aldehydes and in the para position for aromatics (99). 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Most of the vanillic acid was reduced by E. coli containing Car in 2 h to vanillin (80 %) and vanillyl alcohol (20 %). Car does not reduce aldehydes to alcohols. However, E. coli s endogenous aldehyde reductase/dehydrogenase reduces vanillin to vanillyl alcohol. The broad substrate specificity of Car enables the wide application of this biocatalyst to other important applications, such as enantiomeric resolution of isomers such as ibuprofen and the reductions of many other natural and synthetic carboxylic acids. 

KarmaData contains information which the user enters, e.g., QSAR equations, congener set, as well as information about previously studied enzyme-ligand binding complexes. KarmaData contains several classes and subclasses. For example, in KarmaData, there is a class called proteins, a subclass in proteins called dehydrogenase, a particular member of dehydrogenase c led DHFR, and a specific instance of DHFR called chicken (vide ir a). Chicken DHFR contains those attributes which are specific to itself, and inherits properties from units DHFR, dehydrogenase, and proteins. 

Figure 3.1 Amino add side-chain groups involved in binding NAD at the active site of an enzyme. The enzyme is glyceraldehyde dehydrogenase. More than 20 amino acids, the position of which in the primary structure is indicated by the number, counting from the N-terminal amino acid, are involved in the binding. This emphasises the complexity of the binding that is responsible for the specificity of the enzyme for NAD (depicted in bold). The molecular structure of nicotinamide adenine dinucleotide (NAD ) provided in Appendix 3.3.

NADH. These experiments were pioneering with respect to contemporary enzymology, especially with regard to early recognition that coenzymes are held within enzyme active sites in stereochemically preferred ways. One typically utilizes NADH that contains a tritium or deuterium atom in the 4R or 45 position, and the success or failure of substrate deuteration/tritiation indicates the stereochemistry. Westheimer has tabulated the known examples of dehydrogenases that exhibit specificity for a particular face of NADH. Creighton and Murthy have reproduced this tabulation in their comprehensive review on the stereochemistry of enzyme-catalyzed reactions at carbon. 

Preparative Syntheses of Chiral Alcohols using (R)-Specific Alcohol Dehydrogenases 341... 

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