Dehydrogenase enzymes, selected enzyme

Enzyme-coupled ECL enables the selective determination of many clinical analytes that are not in themselves directly electrochemiluminescent, but that can act as substrates for a variety of enzymic reactions. There are two general strategies for ECL the use of dehydrogenase enzymes, which convert NAD+ to NADH, and oxidase enzymes, which produce hydrogen peroxide. 

In the case of horse liver alcohol dehydrogenase, a homodimeric enzyme, Subramanian et al.(202) used the relative phosphorescence of tyrosine and tryptophan to examine the effects of various ternary complexes known to selectively quench the fluorescence of the tryptophans of each subunit. One proposed quenching mechanism is the formation of a ground-state tyrosinate in a ternary complex at neutral pH.(201) This tyrosinate, by being a resonance... 

Incubation of the (1R,2R) diol 25 over a shorter time period of 4 days gave a mixture of the meso cis diol 26 (26%) and the trans diol 25 RR/SS 76 26) showing conversion over to the (1S,2S) diol. As with the previous examples, no conversion was observed when using the S,S diol 25 as a substrate. Although no intermediate a-hydroxyketone was observed for this substrate we proposed the operation of at least two dehydrogenase enzymes, DH-1 and DH-2, catalysing the (i )-selective oxidation and (S)-selective reduction respectively (Scheme 12). Incubation of cis cycloheptan-l,2-diol afforded only the (S)-2-... 

A somewhat different approach to determining the enantiopurity of a sample is based on the idea that an appropriate enzyme selectively processes one enantiomer, giving rise to a UV/visible signal [17]. An example concerns determination of the enantiopurity of chiral secondary alcohols, the (S) enantiomer being oxidized selectively by the alcohol dehydrogenase from Thermoanaerobium sp. The rate of this process can be monitored by a UV/visible plate reader due to the formation of NADPH (absorption at 340 nm), which relates to the quantity of the (S) enandomer present in the mixture. About 4800 ee determinadons are possible per day, accuracy amoundng to 10%. Although the screen was not specifically developed to evaluate chiral alcohols produced by an enzymadc process, it is conceivable that this could be possible after an appropriate extraction process. 

The biochemical characterization of several alcohol dehydrogenases and their exploitation potential demonstrate that these enzymes are most important tools for biochemists. Amino acid sequences of several ADFls are available so far, and alignment studies allow to establish ADH families and to consider their probable evolutionary relationships. For preparative applications, however, particular properties of an enzyme are essential prerequisites, such as enzyme stability and availability, its substrate specificity, or reaction selectivity. Enzymes with NAD as coenzyme are clearly preferred to NADP-dependent ones in practice, because NAD has a significantly higher stability [186-188], a lower price and, is in general, easier to regenerate. 

The commercial availability of various lactate-specific enzymes and the high demand for lactate analysis for clinical and food processing led to the construction of numerous lactate enzyme electrodes (Tkble 8). Depending on the selected enzyme—lactate oxidase (LOD), lactate mono-oxidase (LMOD), or lactate dehydrogenase (LDH)—several mechanistic approaches may be applied ... 

Lactate dehydrogenases represent another important subgroup of carbonyl reductases. They reduce a-oxo acids enantiospecifically to a-hydroxy acids. Both the D- and the L-selective enzymes are available, giving access to both enantiomers of various a-hydroxy acids [39]. 

Fluorescence detection is also inherently more selective than absorbance detection, since both the excitation and emission wavelengths may be chosen to suit a particular reaction product. For example, assays employing dehydrogenase enzymes may monitor NAD+ or nicotinamide adenine dinucleotide phosphate (NADP+) absorbance at 340 nm with reasonable sensitivity and selectivity. However, if excited at 340 nm, the nicotinamide coenzymes fluoresce at 460 nm. Not only do the fluorescence measurements inherently have lower detection limits, but they also provide selectivity against potential interferents that may also absorb at 340 nm but do not emit at 460 nm. 

A low enantiomeric excess of a reduced product can result from the inability of the oxidoreduc-tase to recognize the structural features of the substrate enantio- and diastereoselectively45. By far the more common case is the competition of two or more enzymes with different enantiose-lectivities and Michaelis constants for the substrate. Yeast cells not only contain yeast alcohol dehydrogenase but several enzymes that arc able to catalyze reductions. At least three different enzymes capable of reducing /i-keto esters have been purified, of which two have D- and one has L-selectivity62-65. Yeast alcohol dehydrogenase has a rather narrow selectivity for short-chain alcohols and aldehydes and certainly is not the catalyst in many of the reductions performed with yeasts. 

Ottolina and co-workers47 showed the oxidation of a variety of diones selectively using isolated enzymes. The enzyme system proved quite sensitive to changes in oxidation state and substitution in the substrate. In this instance, glucose dehydrogenase (GDH) was used as the second enzyme system to replenish NADPH. The Baeyer-... 

Trinuclear clusters have been detected in over 20 proteins as well as a number of enzymes, among them aconitase, beef heart succinate-ubiquinone oxidoreductase (120), Escherichia coli nitrate reductase (121), E. coli fumarate reductase (122), and succinate dehydrogenase (123). Selected instances of the occurrence of [3Fe-4S] clusters are listed in Table II. Because of the paramagnetic ground states of both oxidation levels, these clusters can be uniquely identified by a number of spectroscopic techniques. Among these, Mossbauer spectroscopy in applied magnetic fields (124, 128, 132, 141-143) and low temperature MCD spectroscopy (127, 138, 144-146) are decisive. While there are small spectroscopic differences among certain [3Fe-4S] centers, the similarities dominate and support the essential structure 3 for all. In a number of the earlier papers on protein... 

Reactions of oxidoreduction are an example of chiral inversion that takes place by the intermediacy of two opposing metabolic processes. The alcohol/ketone equilibrium mediated by alcohol dehydrogenase enzyme is an abundant reaction. The dehydrogenation of secondary alcohols to a ketone proceeds with substrate stereoselectivity in oxidation, while the hydrogenation of the ketone metabolite is product selective to one face of the carbonyl group. The consequence of the metabolism of the secondary alcohols may involve chiral inversion of this center, which can result in an altered proportion of the two enantiomers or epimers. 

A wide variety of biosensors can be developed by coupling selective dehydrogenase enzymes with the HAD FMH oxidoreductase and bacterial luciferase system. An example is a glutamate biosensor based on the following reaction scheme ... 

The first example of a fiber-optic internal enzyme biosensor has been reported for the determination of ethanol (13). A microporous Teflon membrane is used as the perm-selective membrane for this ethanol sensor. Alcohol dehydrogenase is the enzyme and the following reactions take place in the sensor ... 

Curulli, A., I. Carelli, O. Trischitta, and G. PalleschL 1997. Enzyme electrode probes obtained by electropolymerization of monomers with PMS and selected dehydrogenase enzymes. Talanta 44 (9) 1659. 

Dehydrogenase Reduction of aldehydes and ketones Prelog-selectivity, NADH-enzymes, whole cells Anti-Prelog-enzymes, NADPH-enzymes State of the art, NADH-dependent anti-Prelog enzymes ... 

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