Alcohol dehydrogenase mutations

Horse liver alcohol dehydrogenase, F93W mutant with 1224 also mutated to G,A,V,L. hydride transfer from benzyl alcohol to NAD Heterotetrameric sarcosine oxidase of Arthrobacter sp. 1-IN, proton transfer from adduct of FAD with sarcosine-(CH3) and sarcosine-(CD3)... 

Halpin, C., Holt, K., Chojecki, J., Oliver, D., Chabbert, B., Monties, B., Edwards, K., Barakate, A., and Foxon, G. A., 1998, Brown-midrib maize (bml) - a mutation affecting the cinnamyl alcohol dehydrogenase gene, Plant J. 14 545-553. 

Horse liver alcohol dehydrogenase, F93W mutant with 1224 also mutated to... 

Recently, a controversial debate has arisen about whether the optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of tunneling and thereby accelerate reaction rates [7]. Kinetic isotope effect experiments have indicated that hydrogen tunneling plays an important role in many proton and hydride transfer reactions in enzymes [8, 9]. Enzyme catalysis of horse liver alcohol dehydrogenase may be understood by a model of vibrationally enhanced proton transfer tunneling [10]. Furthermore, the double proton transfer reaction in DNA base pairs has been studied in detail and even been hypothesized as a possible source of spontaneous mutation [11-13]. 

Ex ample 1 The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary alcohol dehydrogenase was overexpressed in Escherichia coli to form more than 10% to total protein11361. The recombinant enzyme was purified by heat treatment and precipitation with aqueous (NH4)2S04 and isolated in 67% yield. Enzymes with mutation(s) around the active site residues were also created to examine the catalytically important zinc binding motif in the proteins. 

Point mutation of enzymes has played an important role in determining those amino acid residues involved in catalytic activities. It has also been used to improve the enantioselectivity of dehydrogenases. For example, even a single point mutation of a secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus can change substantially the enantioselectivity for the reduction of 2-butanone and 2-pentanone as shown in Table 15-6 45l... 

Table 15-6. Control of enantioselectivity by a single mutation (serine-39 to threonine) of the secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 5.

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