Fructose, Schiffs base formation

There are two distinct groups of aldolases. Type I aldolases, found in higher plants and animals, require no metal cofactor and catalyze aldol addition via Schiff base formation between the lysine s-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily in microorganisms and utilize a divalent zinc to activate the electrophiUc component of the reaction. The most studied aldolases are fructose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn2+-containing aldolase from E. coll In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetonephosphate [57-04-5] (DHAP). 

Skeletal muscle and yeast phosphofructokinases will catalyse the phosphorylation of 5-keto-D-fructose-l,6-bisphosphate (9). The latter has been isolated chromatographically and identified by its phosphorus content and the rather doubtful method of acid lability of the phosphate groups. The bis-phosphate is a competitive inhibitor of the reaction between aldolases and fructose-1,6-bisphosphate probably because of Schiff base formation with the enzyme. ... 

The stereochemistry of Schiff base formation between fructose 1,6-bisphosphate and the aldolase from liver has also been addressed (777). Suggestive evidence for the intermediate formation of a (27 )-carbinolamine is based on the observation that BH4 reduction of substrate on the enzyme followed by acid hydrolysis of the protein gives exclusively glucitollysine and not mannitollysine. This indicates that the re face of the ketimine is exposed to solvent, and it implies that OH left from the same direction in other to form the ketimine. On this basis, the e-amino of the lysine must add to the si face of the substrate carbonyl lEq. (33)1 ... 

For class I type enzymes, the (/ia)8-barrel structure of the class I fructose 1,6-bisphosphate aldolase (FruA, vide infra) from rabbit muscle was the first to be uncovered by X-ray crystal-structure analysis [33] this was followed by those from several other species [34-37]. A complex of the aldolase with non-covalently bound substrate DHAP (dihydroxyacetone phosphate) in the active site indicates a trajectory for the substrate traveling towards the nucleophilic Lys229 N [38, 39]. There, the proximity of side-chains Lysl46 and Glul87 is consistent with their participation as proton donors and acceptors in Schiff base formation (A, B) this was further supported by site-directed mutagenesis studies [40]. 

The transaldolase functions primarily to make a useful glycolytic substrate from the sedoheptulose-7-phosphate produced by the first transketolase reaction. This reaction (Figure 23.35) is quite similar to the aldolase reaction of glycolysis, involving formation of a Schiff base intermediate between the sedohep-tulose-7-phosphate and an active-site lysine residue (Figure 23.36). Elimination of the erythrose-4-phosphate product leaves an enamine of dihydroxyacetone, which remains stable at the active site (without imine hydrolysis) until the other substrate comes into position. Attack of the enamine carbanion at the carbonyl carbon of glyceraldehyde-3-phosphate is followed by hydrolysis of the Schiff base (imine) to yield the product fructose-6-phosphate. 

Aldolases such as fructose-1,6-bisphosphate aldolase (FBP-aldolase), a crucial enzyme in glycolysis, catalyze the formation of carbon-carbon bonds, a critical process for the synthesis of complex biological molecules. FBP-aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate (DHAP) and glyceralde-hyde-3-phosphate (G3P) to form fructose-1,6-bisphosphate. There are two classes of aldolases the first, such as the mammalian FBP-aldolase, uses an active-site lysine to form a Schiff base, whereas the second class features an active-site zinc ion to perform the same reaction. Acetoacetate decarboxylase, an example of the second class, catalyzes the decarboxylation of /3-keto acids. A lysine residue is required for good activity of the enzyme the -amine of lysine activates the substrate carbonyl group by forming a Schiff base. 

The first part of the reaction is formation of a protonated Schiff base r)f sedoheptulose 7-phosphate with a lysine residue in the enzyme followed by a retro-aldol cleavage to give an enamine plus erythrose 4-j>hosphatc. Show the structure of the enamine and the mechanism by which it is formed. Hie second part of the reaction is nucleophilic addition of the enamine to glyceraldehyde 3-iihosphate followed by hydrolysis of the Sch ff base to give fructose 6-phosphate. Show the mechanism. 

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