Fructose enzymic catalysis

At the present time, commercial isomerization processes based on enzymic catalysis are predominant, so only brief mention will be made of some of the nonenzymic processes that have been considered for commercialization in the past. Probably the major reasons for the current commercial use of enzymic rather than nonenzymic systems are that the nonenzymic systems so far developed result in products having one or more of the following defects too much ash, color, acid, off-flavor, a content of D-mannose or D-psicose, and high ratios of D-glucose to D-fructose. Probably, further advances in our understanding of the isomerization reaction and the mechanisms of the catalysis will lead to more efficient, nonenzymic processes that could replace the enzymic-isomerization systems now used commercially. 

Enzyme Mechanisms.— Triose phosphate isomerase has been a popular enzyme recently, having been the chief example quoted in two reviews on perfection and efficiency in enzyme catalysis - and the subject of seven successive papers in one issue of Biochemistry including one on the evolution of enzyme function and the development of catalytic efficiency. During glycolysis in muscle, fructose 1,6-bisphos-... 

Enzymatic catalysis is also used in the synthesis of l-"C-labeled D-fructose and o-glucose firom mannitol and glucitol, respectively (Ogren and Langstrom 1998). The preparation of S-adenosyl-L-[ C]methionine (Guegen et al. 1982), [ C]epinephrine (Soussain et al. 1984), and [ C]daunorubicin (Eriks-Fluks et al. 1998) are other examples of enzyme catalysis. The nucleosides [ C]thymidine and [ C]-2 -arabino-2 -fluoro-P-5-methyl-uridine have been prepared by enzymes immobilized on hollow fiber membranes using [ C]formaldehyde as the labeled precursor (Hughes and Jay 1995). 

Yeast contains a number of enzymes, more particularly inyertase and zymase. Invertase catalyses the hydrolysis of sucrose to glucose and fructose (cf. the catalysis of this reaction by acids, p. 369). 

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7-8 illustrates the roles of seven active site residues. Catalysis involves a catalytic triad of one Glu and two His residues and a covalent phos-phohistidyl intermediate. 

Fructose 1,6-diphosphatase hydrolyzes D-fructose 1,6-diphosphate to give D-fructose 6-phosphate and PO . It is a key enzyme in the gluconeo-genesis pathway. Two divalent metal ions (Mg2+, Mn2+, Zn2+, and Co2+) are involved in catalysis. In the enzyme isolated from pork kidney the metal-metal distance accounts to 3.7 A [12]. A reaction mechanism similar to that of protein phosphatase 1 was proposed, but leaving group stabilization by metal coordination of the ester oxygen atom appears to be absent (Figure 6) [12]. 

There are three mechanistic possibilities for catalysis by two-metal ion sites (Fig. 10). The first of these is the classic two-metal ion catalysis in which one metal plays the dominant role in activating the substrate toward nucleophilic attack, while the other metal ion furnishes the bound hydroxide as the nucleophile (Fig. 10 a). Upon substrate binding, the previously bridged hydroxide shifts to coordinate predominately with one metal ion. Enzymes believed to function through such a mechanism include a purple acid phosphatase [79], DNA polymerase I [80], inositol monophosphatase [81],fructose-1,6-bisphosphatase [82], Bam HI [83], and ribozymes [63]. 

If a very low molar concentration of enzyme is present, and a large excess of nonradioactive fructose is added, the enzyme will catalyze no net reaction but will change back and forth repeatedly between the free enzyme and glucosyl enzyme. Each time, in the reverse reaction, it will make use primarily of unlabeled fructose. The net effect will be catalysis of a sucrose-fructose exchange ... 

Mechanism of the interconversion of glucose-6-phosphate and fructose-6-phosphate. Loss of a proton from the oxygen attached to C-2 of the intermediate enediol leads to fructose-6-phosphate. A and B represent catalytic groups on the enzyme. It is not always known what specific groups are involved in a catalysis. In this case the HA group originates from a glutamate on the enzyme. 

Primary amine catalysis (usually involving a lysine residue) has been recognised to play an important role in various enzyme-catalysed reactions. Examples are the conversion of acetoacetate to acetone catalysed by acetoacetate decarboxylase, the condensation of two molecules of S-aminolevulinic acid catalysed by -aminolevulinic deshydratase during the biosynthesis of porphyrins, and the reversible aldol condensation of dihydroxy-acetone phosphate with glyceraldehyde which in the presence of aldolase yields fructose-1-phosphate (64) (For reviews see, for example, Snell and Di Mari,... 

G3P) and D-sedoheptulose 7-P as illustrated in Scheme 5.53. In addition D-erythrose 4-phosphate can function as the ketol acceptor thus producing D-fructose-6-P and G3P (Scheme 5.53). The enzyme relies on two cofactors for activity — thiamin pyrophosphate (TPP) and Mg2+—and utilizes the nucleophilic catalysis mechanism outlined in (Scheme 5.54).83 When TPP is used as a cofactor for nucleophilic catalysis, an activated aldehyde intermediate is formed. This intermediate functions as a nucleophile, and thus TK employs a strategy that is similar to the umpolung strategy exploited in synthetic organic chemistry. 

Although TA from yeast is commercially available, it has rarely been used in organic synthesis applications, and no detailed study of substrate specificity has yet been performed. This is presumably due to high enzyme cost and also since the reaction equilibrium is near unity, resulting in the formation of a 50 50 mixture of products. In addition the stereochemistry accessible by TA catalysis matches that of FruA DHAP-dependent aldolase and the latter is a more convenient system to work with. In one application, TA was used in the synthesis D-fructose from starch.113 The aldol moiety was transferred from Fru 6-P to D-glyceraldehyde in the final step of this multi-enzyme synthesis of D-fructose (Scheme 5.60). This process was developed because the authors could not identify a phosphatase that was specific for fructose 6-phosphate and TA offered an elegant method to bypass the need for phosphatase treatment. 

It may be noted that simple alkaline-catalyzed isomerization of glucose to fructose is possible, but gives rise to serious lactic acid and coloured by-product formation. Alkaline catalysis, however, is still applied for the conversion of lactose to lactulose, used in treatment of constipation and PSE. The reason is that no enzyme has been found that is able to isomerize the glucose unit of lactose into a fructose moiety. As a consequence, a low conversion is applied or borate is used as a protecting group. In the latter case extra separation and recycle steps are required. 

Aldolase is an example of an enzyme that uses electrophilic covalent catalysis. The amine of an active site lysine forms an imine (Section 10.5.2) with the carbonyl of fructose-1,6-bisphosphate. This more reactive imine electron sink allows a reverse aldol reaction to occur via the less basic enamine rather than the more basic enolate ion. Tautomerization of the resulting enamine to an imine, then hydrolysis, releases DHAP and returns the enzyme active site lysine to the free anime, ready for the next cycle. 

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