2-Butanone enzymes

In general, pyruvate decarboxylase (EC 4.1.1.1) catalyzes the decarboxylation of a 2-oxocar-boxylic acid to give the corresponding aldehyde6. Using pyruvic acid, the intermediately formed enzyme-substrate complex can add an acetyl unit to acetaldehyde already present in the reaction mixture, to give optically active acetoin (l-hydroxy-2-butanone)4 26. Although the formation of... 

On the other hand, as mentioned in the preceding subsection, a preparative-scale enzymic synthesis of 1-deoxy-D-r/ireo-pentulose can be achieved, according to Reaction 1, in the presence of an extract of B. pumilus. Obviously, this raises the question of the relevance of Eq. 1 to the production of the pentulose in microorganisms. Acetoin in Reaction 1 could be replaced by 3-hydroxy-3-methyl-2-butanone (then the by-product is acetone). More interestingly, it can be also replaced by pyruvate, then the pentulose is synthesized according to Reaction 3 ... 

In contrast to Mori s synthesis, Pawar and Chattapadhyay used enzymatically controlled enantiomeric separation as the final step [300]. Butanone H was converted into 3-methylpent-l-en-3-ol I. Reaction with trimethyl orthoacetate and subsequent Claisen-orthoester rearrangement yielded ethyl (E)-5-methyl-hept-4-enoate K. Transformation of K into the aldehyde L, followed by reaction with ethylmagnesium bromide furnished racemic ( )-7-methylnon-6-ene-3-ol M. Its enzyme-catalysed enantioselective transesterification using vinylacetate and lipase from Penicillium or Pseudomonas directly afforded 157, while its enantiomer was obtained from the separated alcohol by standard acetylation. 

The two established Hnls, those from L. usitatissimum and P. amygdalus, have found biocatalytic applications for the production of (i )-cyanohydrins. The former of these Hnls is the least widely applied, the natural substrates being acetone cyanohydrin or (i )-2-butanone cyanohydrin (Table 1) [28]. Although an improved procedure for the purification of this enzyme has been reported [27] it is still only available in limited quantities (from 100 g of seedlings approximately 350 U of enzyme are obtained). It was found that this enzyme transforms a range of aliphatic aldehyde and ketone substrates [27], the latter of which included five-membered cyclic (e.g. 2-methylcyclopentanone) and chlorinated ketone substrates. In contrast, attempts to transform substituted cyclohexanones and 3-methylcyclopentanone failed and it was even found that benzaldehyde deactivated the enzyme. 

Traiger GJ, Bruckner JV, Jiang WD, et al. 1989. Effect of 2-butanol and 2- butanone on rat hepatic ultrastructure and drug metabolizing enzyme activity. J Toxicol Environ Flealth 28 235-248. 

Another base-catalyzed reaction is the addition of enolate anions derived from ketones to the 4 position of the pyridine nucleotides (Eq. 15-19). The adducts undergo ring closure and in the presence of oxygen are converted slowly to fluorescent materials. While forming the basis for a useful analytical method for determination of NAD+ (using 2-butanone), these reactions also have created a troublesome enzyme inhibitor from traces of acetone present in commercial NADH.132... 

Figure 9. Photoreduction of 2-butanone using two coupled enzymes and the NADPH regeneration cycle.

A major aspect to consider in such enzyme-catalyzed photochemical systems is the stability of enzymes in the artificial chemical environments. Table III summarizes the turnover (TN) numbers for the different enzymes and cofactors involved in the reduction of 2-butanone. It is evident that the enzymes exhibit high stability... 

By using water-soluble organic solvents, usually referred to as precipitants, the reaction can be directed to produce only one cyclodextrin. In the presence of toluene, the enzyme from B. macerans produces only (3-cyclodextrin and linear starch chains. Beta-cyclodextrin can be separated from the soluble starch chains and recovered as a precipitated toluene complex. In the presence of decanol, a-cyclodextrin is the only cyclodextrin produced using the enzyme from B. macerans.21,22 Precipitants such as large cyclic compounds similar to musk oil23 or a-naphthol and methyl ethyl ketone (butanone)24 can be used to produce 8-cyclodextrin. 

Figure 13. Continuous formation of (S )-4-phenyl-2-butanol from 4-phenyl-2-butanone using the electrochemical enzyme membrane reactor under indirect electrochemical NADH regeneration with a high-molecular-weight rhodium catalyst [26,29,30,65].

The stereochemistry of the acetoacetate decarboxylase catalyzed reaction was also investigated. It was first demonstrated that the enzyme selectively exchanged one of two methylene protons of butanone with deuterons . Subsequently, it was shown using substrates 17 and 18 that the reaction proceeds with retention of configuration giving 19 and (equation 12), as appears to be the case in the pyridoxal catalyzed... 

An enzymic counterpart of these complex base-catalysed rearrangements of sugars may be the reaction catalysed by 4-phospho-3,4-dihydroxy-2-butanone synthetase. The enzyme catalyses the formation of the eponymous intermediate in secondary metabolism from ribulose 5-phosphate. Labelling studies indicated that C1-C3 of the substrate became C1-C3 of the product, that H3 of the substrate derived from solvent and that C4 was lost as formate. X-ray crystal structures of the native enzyme and a partly active mutant in complex with the substrate are available. The active site of the enzyme from Met ha-nococcus jannaschii contains two metals, which can be any divalent cations of the approximate radius of Mg " or Mn ", the two usually observed. Their disposition is very reminiscent of those in the hydride transfer aldose-ketose isomerases, but also to ribulose-5-phosphate carboxylase, which works by an enolisation mechanism, so the enolisation route suggested by Steinbacher et al. is repeated in Figure 6.14, as is the Bilik-type alkyl group shift, for which an equivalent reverse aldol-aldol mechanism cannot be written. 

Two approaches were studied to obtain (R)-l,3-BDO. The first was based on an enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone, and the second was based on enantioselective oxidation of the undesirable (S)-l,3-BDO in the racemate. As a result of screening for yeasts, fungi, and bacteria, the enzymatic resolution of racemic 1,3-BDO by Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-l,3-BDO with high enantiomeric excess and yield. 

In the past ten years, there has been developed a series of enzyme inhibitors that combine the features of an alkylating agent with specificity for the active site of an enzyme, thus permitting alkylation and identification of a group at or near the active center of an enzyme, or a particular enzyme to be specifically inactivated. Thus a l-chloro-4-phenyl-3-p-toluenesulfonamido-2-butanone ( W-p-tolylsulfonylphenylalanine chloro-methyl ketone ) inactivates chymotrypsin (which cleaves a peptide bond adjacent to an aromatic residue), and 7-amino-l-chloro-3-p-toluene-sulfonamido-2-heptanone ( a-iV-p-tolylsulfonyllysine chloromethyl ketone ) inhibits trypsin (which cleaves a peptide bond adjacent to lysine. In both cases, a histidine residue at the active site is alkylated, and neither inhibitor will inhibit the other enzyme at low concentrations. 

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