Dehydrogenase stereospecificity

We can thus deduce that alcohol dehydrogenase stereospecifically removes the pro-R hydrogen from the prochiral methylene. 

S. Mostad and A. Glasfeld,/. Chem. Educ. 70, 504-506 (1993). Using High Field NMR to Determine Dehydrogenase Stereospecificity with Respect to NADH. ... 

C Mathews, K van Holde, and K Ahern, Biochemistry, 3rd ed (2000), Benjamin/ Cummings (San Francisco), pp 202-208 Introduction to spectroscopic methods F Mirabella, Modem Techniques in Applied Molecular Spectroscopy (1998), John Wiley Sons (New York) Theory and applications in all areas of spectroscopy S Mostad and A Glasfeld,/ Chem. Educ. 70, 504-506 (1993) Using High Field NMR to Determine Dehydrogenase Stereospecificity with Respect to NADH. ... 

Davis J, Jones JB (1979) Enzymes in organic synthesis. 16. Heterocyclic ketones as substrates of horse liver alcohol dehydrogenase. Stereospecific reductions of 2-substituted tetrahydropyran-4-ones. J Am Chem Soc 101 5405-5410... 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

As noted in Chapter 18, the enzymes that require nicotinamide coenzymes are stereospecific and transfer hydride to either the pro-i or the pro-S positions selectively. The table (facing page) lists the preferences of several dehydrogenases. 

NAD (P) " -dependent enzymes are stereospecific. Malate dehydrogenase, for example, transfers a hydride to die pro-/ position of NADH, whereas glyceraldehyde-3-phosphate dehydrogenase transfers a hydride to die pro-5 position of the nicotinamide. Alcohol dehydrogenase removes a hydride from the pro-i position of edianol and transfers it to die pro-i position of NADH. 

The stereospecificity of hydride transfer in dehydrogenases is a consequence of the asymmetric nature of die acUve site. 

Figure 11-4. Mechanism of oxidation and reduction of nicotinamide coenzymes. There is stereospecificity about position 4 of nicotinamide when it is reduced by a substrate AHj. One of the hydrogen atoms is removed from the substrate as a hydrogen nucleus with two electrons (hydride ion, H ) and is transferred to the 4 position, where it may be attached in either the A or the B position according to the specificity determined by the particular dehydrogenase catalyzing the reaction. The remaining hydrogen of the hydrogen pair removed from the substrate remains free as a hydrogen ion.

There are some very interesting questions of stereospecificity posed by the structure and mode of operation of multienzyme complexes. Reed and Cox 35> have summarized available information on the pyruvate and a-ketoglutarate dehydrogenase complexes, and the fatty add synthetase. The mechanism of synthesis of the peptide antibiotics likewise presents interesting stereochemical problems 36>. 

The liver alcohol dehydrogenase mentioned in the preceding section has the same pro-R stereospecificity for NAD and ethanol as yeast alcohol dehydrogenase. Furthermore, the oxidation of ethanol by a microsomal oxidizing system, or by catalase and H2O2, likewise proceeds with pro-R stereospecificity for the ethanol77>. The catalase-H2C>2 system is so very different, however, from the pyridine nucleotide dehydrogenase, that one wonders whether the similarity in stereospecificity for ethanol is fortuitous. 

The stereospecificity of hydrogen transfer for estradiol-17 and estradiol-17(3 dehydrogenases has been examined by George et a/.84>. These enzymes are both present in chicken liver, and have substrates which differ only in the chirality of their substituents at C—17. Both of these enzymes were shown to use the 4-pro-S or 4B proton of the NADPH. Since the steroid is a bulky substrate, the authors argue that the steric fit between pyridine nucleotide and steroid cannot be as important as the role played by the enzyme in directing the fit. This paper contains an interesting summary of other recent work on the stereospecificity of pyridine nucleotide dependent-steroid dehydrogenases. 

It should be noted, in this connection, that there are pyridine nucleotide dehydrogenases which catalyze redox reactions which must occur in two steps. Hydroxymethylglutaryl CoA reductase (discussed on p. 51) is one example. Another is uridine diphosphate-glucose dehydrogenase, which catalyzes the oxidation of the C—6 of the glucose (i.e., a primary alcohol) to a carboxyl group. In both cases, there are two molecules of pyridine nucleotide required, and the overall reactions are essentially irreversible. The former enzyme, with A stereospecificity for the pyridine nucleotide, catalyzes the reduction of an acyl-CoA group... 

In an earlier spectrophotometric study of this enzyme, a red shift of the reduced nicotinamide absorbance had been noted in the difference spectrum of the binding of reduced coenzyme to the purified protein. Fisher et al.92> had pointed out that this is characteristic of most B-stereo-specific dehydrogenases, so Biellman et al. have made a successful prediction for Fisher. Fisher s suggestion that the supernatant and mitochondrial forms of malate dehydrogenase have different stereospecificities for NAD+ has not been substantiated, however 89>. 

Liver cells contain two different but closely related enzymes glycerol phosphate dehydrogenase which is specific for NAD, and acylglycerol phosphate dehydrogenase, which is NADP specific. Both enzymes have B stereospecificity for the pyridine nucleotide 93. They apparently have different metabolic functions. 

The generalization that the same dehydrogenase has the same stereospecificity, no matter what the source of the enzyme, has been tested now particularly well for malic and lactic dehydrogenases. In fact, one can venture a guess, that pyridine nucleotide dehydrogenases which oxidize a-hydroxycarb oxylic acids at the a-position, all have A stereospecificity for the pyridine nucleotide, regardless of their stereo-specificity for the substrate. Biellman and Rosenheimer 88> have assembled the data. One can add liver malic enzyme 90> to their list. 

Betz, G., Warren, J. C. Reaction mechanism and stereospecificity of 20 jS-hydroxysteroid dehydrogenase. Arch. Biochem, Biophys. 128, 745—752 (1968). 

Fisher, H. F., Adija, D. L., Cross, D. G. Dehydrogenase-reduced coenzyme difference spectra, their resolution and relationship to the stereospecificity of hydrogen transfer. Biochemistry 8, 4424—4430 (1969). 

The chiral compounds (/ )- and (5)-bis(trifluoromethyl)phenylethanol are particularly useful synthetic intermediates for the pharmaceutical industry, as the alcohol functionality can be easily transformed without a loss of stereospecificity and biological activity, and the trifluoromethyl functionalities slow the degradation of the compound by human metabolism. A very efficient process was recently demonstrated for the production of the (5)-enantiomer at >99% ee through ketone reduction catalyzed by the commercially available isolated alcohol dehydrogenase enzyme from Rhodococcus erythropolis (Figure 9.1). The (7 )-enantiomer could be generated at >99% ee as well using the isolated ketone reductase enzyme KRED-101. 

Bartoschek, S., Buurman, G., Thauer, R. K., Geierstanger, B. H., Weyrauch, J. P., Griesinger, C., Nilges, M., Hutter, M. C., Helms, V. (2001) Re-face stereospecificity of methylenetetrahy-dromethanopterin dehydrogenases and methylenetetrahydrofolate dehydrogenases is predetermined by intrinsic properties of the substrate. Chem Bio Chem, in press. 

Several microbial alcohol oxidoreductases can catalyze the stereoselective oxidation of GLD. Quinohaemoprotein ethanol dehydrogenase of Acetobacter pasteurianus is able to oxidize stereospecifically (S)-GLD to (l )-glycidic acid in racemic GLD [41,42 ]. When washed cells of A. pasteurianus were incubated with 4.8 mg ml" of racemic GLD, (1 )-GLD was obtained, with an optical purity of 99.5% e. e. and 64% conversion. 

Fig. 6. Outline of the stereospecific reduction of CAAE by aldehyde reductase (AR) with glucose dehydrogenase (GDH) as the cofactor regenerator in an organic solvent-water two-phase system...

Dihydroflavonol 4-reductase (DFR EC 1.1.1.219) is a member of the short-chain dehydrogenase/reductase family and catalyzes the stereospecific conversion of (+)-(2R,3R)-dihydroflavonols to the corresponding (2R,3S,4S) flavan-3,4-cw-diols (leucoanthocyanidins), with NADPH as a required cofactor. The enzyme activity was first identified in cell suspension cultures of Douglas fir (Pseudotsuga menziesii) and was shown to be related to the accumulation of flavan-3-ols and proanthocyanidins [96]. Leucoanthocyanidins and DFR were later shown to be required for anthocyanidin formation by complementation of Matthiola incana mutants blocked between dihydroflavonol and anthocyanidin biosynthesis [97, 98], DFR has been purified to apparent homogeneity and biochemically analyzed from flower buds of Dahlia variabilis [99]. DFR was shown to accept different substrates depending on the plant species from which it was isolated (reviewed in 100). 

Moinuddin SGA, Youn B, Bedgar DL et al (2006) Secoisolariciresinol dehydrogenase mode of catalysis and stereospecificity of hydride transfer in Podophyllum peltatum. Org Biol Chem 4 808-816... 

The stereospecificity depends upon the enzyme in question. Let us consider the enzyme alcohol dehydrogenase, which is involved in the ethanol to acetaldehyde interconversion. It has been deduced that the hydrogen transferred from ethanol is directed to the Re face of NAD+, giving NADH with the AR configuration, hi the reverse reaction, it is the 4-pro-R hydrogen of NADH that is transferred to acetaldehyde. 

In contrast, an enzymic reduction utilizing NADH will be executed stereospecifically, with hydride attaching to one particular face of the planar carbonyl. Which face is attacked depends upon the individual enzyme involved. For example, reduction of pymvic acid to lactic acid in vertebrate muscle occurs via attack of hydride from the Re face (see Section 3.4.7), and produces the single enantiomer (S )-lactic acid. Hydride addition onto the alternative Si face is a feature of some microbial dehydrogenase enzymes. 

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