Spinach transketolase

Subsequent studies196 on crystalline transketolase have revealed the presence of a contaminating enzyme termed pentulose 5-phosphate waldenase (or epimerase) the presence of which had led to the erroneous conclusion that d-erythro-pentulose 5-phosphate was the substrate for transketolase. d-erythro-Pentulose 5-phosphate is virtually unattacked by transketolase prepared from spinach or liver. In subsequent discussions of experiments involving the use of transketolase, in this article, the enzymic reactions must be viewed as the result of action of transketolase and pentulose 5-phosphate waldenase (epimerase). 

An impediment to a wider use of transketolases, however, was the lack of the catalyst itself, which had to be tediously purified from sources such as baker s yeast or spinach leaves [4]. With the advent of recombinant DNA technologies. 

Aldehydes lacking an OH group at C2 are also transformed by transketolase, leading to a 3S configuration of the hydroxyl group in the deoxyketose product [7a, 9] albeit with a significantly lower rate than with the hydroxylated acceptors [6b, 10. In contrast to the transketolases from spinach and yeast [9, no conversion of aromatic aldehydes, e.g., benzaldehyde or hydroxybenzaldehydes, could be detected with purified E. coli transketolase [6b]. 

For synthetic purposes the E. coli transketolase has a certain advantage over the enzymes from spinach and yeast, because the conversion of a-hydroxypyru-vate with a rate of 60 U (mg of protein) [6b] is significantly higher than the rates of 2 U mg" and 9 U mg reported for the spinach and yeast enzymes [9, 11],... 

Transketolase from common yeast (Saccharomyces cerevisiae) is commercially available, but it is possible to work with a partially purified enzyme, isolated with little expense from spinach leaves.54 Transketolase catalyzes the transfer of a hydroxyacetyl group, reversibly from a ketose phosphate, or irreversibly from hydroxypyruvate to an acceptor aldose, phosphorylated or not.55 It requires thiamine pyrophosphate as a coenzyme, but only in catalytic amounts. In all the cases listed in Table V, the new chiral center, C-3 of the ketose, has the l-glycero configuration. 

The first stage, involving the transfer of active glycolaldehyde, can be accomplished in the laboratory by use of spinach or rat-liver transketolase, and the products isolated and characterized as the barium salt and 2,7-anhydride, respectively. The second stage is catalyzed by liver or yeast transaldolase and is believed to involve the enzymic transfer of a 1,3-di-hydroxy-2-propanone residue sedoheptulose 7-phosphate and D-fructose... 

An enzyme closely related to the aldolases is transketolase. The enzyme is commercially available (from baker s yeast) and can also be obtained from spinach leaves. Transketolase catalyses the stereospecific synthesis of C—C bonds using aldehydes as the electrophiles, with a suitable 2-carbon ketol donor [e.g. hydroxypyruvate (9)] as the nucleophile (Scheme 5.11). The use of hydroxypyruvate ensures that the reaction goes to completion, since carbon dioxide is evolved as the by-product, and hence the reaction is irreversible. In addition, both magnesium ions and catalytic thiamine pyrophosphate are required as co-factors. 

Transketolases from various sources have been shown to possess a broad acceptor spectrum yielding products with complete (5)-stereospecificity for the newly formed stereocenter [1505]. Generic aldehydes are usually converted with full stereocontrol and even a,(3-unsaturated aldehydes are accepted to some degree. However, hydroxylated aldehydes show enhanced rates by mimicking the natural substrate [1499]. Transketolases can be obtained from yeast [1506] and spinach [1507] and their overexpression has opened the way for large-scale production [1508, 1509]. 

The action of an enzyme, transketolase, from rat liver or from spinach, on pentose 5-phosphate results in the formation of a triose phosphate and an ester of sedoheptulose, which is presumably sedoheptulose 7-phosphate. The heptulose has been identified by the preparation of sedoheptulosan tetrabenzoate (227). 

Racker 56) demonstrated the synthesis of carbohydrates from carbon dioxide and hydrogen in a cell-free system by bringing together many of the enzymes listed in Table IV. A spinach fraction furnished the phospho-pentokinase, carboxydismutase, phosphopentosisomerase, transketolase, transaldolase, and hexose diphosphatase. To this fraction were added the other enzymes, DPN+, ATP, and a hydrogenase preparation. The hydro-genase enzyme furnished DPNH in the presence of hydrogen. When this mixture was incubated at 25° for 60 minutes, the synthesis of fructose 6-phosphate could be demonstrated. 

A most important clue to the nature of the steps between pentose phosphate and hexosemonophosphate, and thus to the role of the pentose phosphate pathway in photosynthesis, came from our discovery in 1953 of sedoheptulose 7-phosphate as the first product formed from pentose phosphate. The enzyme transketolase had been purified from rat liver and spinach in my laboratory and crystallized from yeast by Racker and his coworkers and the two laboratories simultaneously discovered that this enzyme contained thiamine pyrophosphate as its functional group, f Isotope studies in my laboratory showed that sedoheptulose was formed by the transfer of a C2 group ( active glycolaldehyde ) from one molecule of pentose phosphate to another, and that the reaction was fully reversible thus sedoheptulose 7-phosphate was also a Ca-donor. In addition, Racker s laboratory made the important finding that fructose 6-phosphate would also yield active glycolaldehyde, and Arturo Bonslgnore and his coworkers discovered that rat liver extracts catalyzed the rapid non-oxidative conversion of hexose phosphate to sedoheptulose phosphate. ... 

Transketolase from spinach leaves has been used in the stmeoq)ecific condensation of hydroxypyruvic acid with a variety of aldehydes, including free sugars, which gives ketoses with (5) configuration at the new chiral centre. An example is given in Scheme 1. ... 

Pentose Phosphate Metabolism. The interconversion of ribose-5-phosphate and ribulose-5-phosphate is catalyzed by phosphoribo-isomerase which Axelrod et al. (20) have shown to occur in pea and spinach leaves. Axelrod and Jang (22) have purified this enzyme 380-fold from alfalfa leaves. The further transformation of ribulose 5-phosphate to xylulose-5-phosphate is catalyzed by phosphoketo-pentose epimerase, which occurs in green leaves (J. Hurwitz and B. L. Horecker, unpublished observation). Transketolase, which reversibly transforms ribose-5-phosphate and xylulose-5-phosphate to sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate, and... 

Transketolase catalyzes the transfer of a Cj fragment from a suitable ketol donor to an aldehyde acceptor. The enzyme has been purified from liver and spinach by Horecker et al. (183) and crystallized from yeast by de la Haba et al. (91). All three enzyme preparations require thiamine pyrophosphate as coenzyme although the precise function of this prosthetic group remains unknown. Presumably a glycolaldehyde-thiamine pyrophosphate-enzyme complex is formed this has been termed active glycolaldehyde (298). A number of phosphate esters have been shown to act as active glycolaldehyde donors, including xylulose-5-phosphate (175,324),... 

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