Epimerase Reactions

There is good evidence for the existence of the 4-oxo intermediate and this means that the reversal of configuration does not arise from a bimolecular nucleophilic substitution (or Sn2 reaction), but from a unimolecular (Snl) reaction forming a carbonium ion. Hence the stereochemistry must reflect 

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FIGURE 23.31 The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3. 

D-Methylmalonyl-CoA, the product of this reaction, is converted to the L-isomer by methylmalonyl-CoA epunerase (Figure 24.19). (This enzyme has often and incorrectly been called methylmalonyl-CoA racemase. It is not a racemase because the CoA moiety contains five other asymmetric centers.) The epimerase reaction also appears to involve a carbanion at the a-position (Figure 24.20). The reaction is readily reversible and involves a reversible dissociation of the acidic a-proton. The L-isomer is the substrate for methylmalonyl-CoA mutase. Methylmalonyl-CoA epimerase is an impressive catalyst. The for the proton that must dissociate to initiate this reaction is approximately 21 If binding of a proton to the a-anion is diffusion-limited, with = 10 M sec then the initial proton dissociation must be rate-limiting, and the rate constant must be... 

Maitra, U. S., Ankel, H. The intermediate in the uridine diphosphate galactose 4-epimerase reaction. J. Biol. Chem. 248, 1477—1479 (1973). 

Fig. 2. —Possible Mechanisms for the Uridine 5 -(cr-D-Glucopyranosyl Pyrophosphate) 4 -Epimerase Reaction. [E and E denote different conformers of the enzyme.]...

That secretion of 2-acetamidoglucal, which is known to be an intermediate in the complex, 2-epimerase reaction, but cannot be isolated under normal conditions, may point to another defect in this enzyme that may be independent of the presence or absence of a feedback-inhibition receptor site for CMP-Neu5Ac has been discussed.233 An excess of 2-acetamidoglucal may be converted spontaneously into GlcNAc and this, enzymically, into ManNAc both compounds are secreted in the urine of the sialuria patient. As a consequence of these reactions, the cellular concentration of ManNAc may increase to a level leading to the synthesis of additional Neu5Ac from this compound by the action of acylneuraminate pyruvate-lyase. All of these reactions, and known or theoretical interactions, were summarized in Fig. 3 of Ref. 233. 

Few examples of epimerases exist for the synthesis of chiral fine chemicals. One example is the epimerase-catalyzed conversion of /V-acctylglucosaminc to /V-acetyhnannosarninc.288 Another epimerase reaction is used for the preparation of the synthetically useful inositols 289 D-myo-inositol (59) is converted into ij-r ///ro-inositol (60) by the enzyme D-myo-inositol 1-epimerase (Scheme 19.39), which has been cloned into E. coli from an Agrobacterium sp.290... 

By analogy to results obtained in studies on the biosynthesis of dTDP-L-rhamnose,18 dTDP-6-deoxy-L-talose,17 and GDP-L-fucose,41 it seems very likely that dTDP-6-deoxy-L-/yxo-hexos-4-ulose (21), formed by the 3,5-epimerase reaction, remains enzyme-bound. 

Epimerization at C-2 of a sugar nucleotide has been described. An enzyme from Escherichia coli catalyzes the epimerization of UDP-2-acetamido-2-deoxy-D-glucose to UDP-2-acetamido-2-deoxy-D-mannose.59 Such an epimerization, together with the 3,5-epimerase reaction of a 4-ketose and stereospecific reduction at C-4, could lead to inversion at all of the chiral centers of, for instance, 2-amino-2-deoxy-D-glucose. 

In enolase, the substrate, 2-phosphoglycerate (2-PGA) is coordinated to two Mg ions, one of which is liganded to the three conserved carboxylate residues (Asp 246, Glu 295, and Asp 320). Currently, more than 600 enolase sequences have been identified in the databases, and aU are thought to be isofunctional, catalyzing the conversion of 2-PGA to phosphoenolpyruvate. In the MLE subclass of the superfamily, at least three reactions are known to be catalysed — in addition to the lactonisation of muconate, succinylbenzoate synthase, and L-Ala-D/L-Glu epimerase reactions are observed within the 300 members. The MR subclass catalyses at least five reactions, mandelate racemisation and 4 sugar dehydratases. As in the MLE subclass, of the 400 members identified, only 50% of these are functionally assigned. 

Figure 1. Epimerase reactions for the formation of nucleotide sugar donors.

Figure 6 The chemical mechanism of the C5-mannuronan epimerase reaction. The exchange of solvent deuterium into the product is indicated. Note the conformation change in the polymeric substrate that is induced by the epimerization reaction.

It is interesting that the reaction epimerizes 75% of the M residues, but not more. No solvent deuterium incorporation into M residues during the epimerization reaction could be detected, which indicates that the proton abstraction that is the necessary first chemical step in the reaction is irreversible. However, the enzyme does not epimerize all of the M residues in the substrate. The equilibrium constant for epimerizations is usually close to one for reactions that involve simple substrates, so one might expect the epimerase reaction to reach equilibrium when the M content of the alginate is equal to the G content. The fact that the reaction occurs on a polymeric substrate made up of chiral monomers complicates considerations of the energetics of the reaction somewhat. Nonetheless, it is difficult to reconcile the apparent irreversibility of proton abstraction with the 1 3 M G ratio in the product, unless one proposes that the conformation of the polymer becomes such that it can no longer bind to the enzyme. 

One of the interesting features of the epimerase reaction is the potential for processivity. After the enzyme catalyzes the epimerization of one residue in the polymeric substrate, it could dissociate and rebind at another location, or it could translocate to another residue without releasing the polymer. To investigate whether AlgE operated in a processive manner, the composition of the product as a function of the extent of epimerization of the polymer was characterized. The experimental results, determined by H and C NMR spectroscopy,... 

Alginate lyase catalyzes cleavage of the glycosidic bond through /3-elimination. The similarity between the lyase reaction and the C-5 mannuronan epimerase reaction has been noted. Interestingly, the proteins share little sequence homology. 

It has been suggested by de Robichon-Szulmajster tj at uridine 5-(a-D-glucopyranosyl pyrophosphate) assumes, without strain, a folded conformation in which the pyrimidine residue is located in close proximity to the a-D-glucopyranosyl moiety and actually participates in the epimerase reaction. The ability of glycosyl nucleotides to assume such a conformation may help to account for the large number of interconversions which these compounds are able to undergo. 

The most likely mechanism of the reaction is shown in Figure 2.1. It involves two half-reactions , with the formation of a 4-keto sugar intermediate. In the first half-reaction this is precisely reversed, except that the direction of addition of hydrogen is such as to reverse configuration at C-4. Each half-reaction shows some formal resemblance to the Meerwein-Ponndorf reduction of ketones to secondary alcohols, in that the second halfreaction is in the same direction as the Meerwein-Ponndorf reaction, while the first is its reverse. In the Meerwein-Ponndorf reaction the hydride ion is the reducing species and is derived from aluminium isopropoxide in 4-epimerase reactions effective H is derived from NADH, which becomes NAD. ... 

Pyrophosphate Uridyl Transferase. The over-all epimerase reaction is composed of three individual reactions. The sugar phosphates are not converted directly into each other, but are first incorporated into uridine nucleotides. Epimerization occurs in the hexose component of the UDP-sugar, which then is cleaved to yield the epimerized sugar phosphate. The synthesis of both UDPG and the corresponding galactose derivative, UDPGal, are catalyzed by enzymes called uridyl transferases. A pyrophosphate (PP) uridyl transferase has been studied in yeast and in several mammalian tissues. It catalyzes the reaction... 

Since the aspects of galactose metabolism which have been touched upon briefly above are covered in many reviews (see above), the following discussion will be concaned mainly with some recent work on the mechanism of the UDP-glucose 4-epimerase reaction. 

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