6.4.2—
The Glyoxyllate Cycle

The acetyl-CoA derived from fatty acid breakdown in germinating seeds could be consumed by the TCA cycle, as indicated by the experiments of Stumpf and Barber (1956). However this would not result in the net formation of a glucose precursor since for each molecule of acetyl-CoA consumed two molecules of carbon dioxide would be produced. Furthermore, it was known at the time that little of the fatty acid was oxidized to CO₂ but instead contributed to a net synthesis of sugars.

The problem of how acetyl-CoA was converted to a glucose precursor was solved by the discovery of the glyoxyllate cycle, by Kornberg and Krebs in 1957. This cycle represents a modification of the tricarboxylic acid cycle in which two molecules of acetyl-CoA are consumed and a molecule of succinic acid is formed (Fig. 6.4). Five enzymes are involved in this process three of which,

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Figure 6.4
The glyoxyllate cycle.

citrate synthetase, aconitase, and malic dehydrogenase are components of the TCA cycle. The remaining two enzymes, the key enzymes of the glyoxyllate cycle, are isocitric lyase (isocitratase) and malate synthetase. The first reaction of the glyoxyllate cycle, catalyzed by citrate synthetase, is the condensation of oxaloacetate and acetyl-CoA to form citrate, which is then converted to isocitrate by the action of aconitase. The next reaction, unique to this cycle, is the cleavage of isocitrate into succinate and glyoxyllate catalyzed by isocitrate lyase. One of the products of this reaction, glyoxyllate, is then condensed with a second molecule of acetyl-CoA under the catalytic action of malate synthetase, to produce one molecule of malate. Malate is then oxidized by malate dehydrogenase to oxaloacetate with the concomitant reduction of NAD⁺ . The overall equation for the cycle is therefore:

Succinate produced by the glyoxyllate cycle can then be converted to hexose by conversion to oxaloacetate, by the action of succinic dehydrogenase and fumarase. The oxaloacetate is converted to phosphoenolpyruvate, a reaction catalised by phosphoenolpyruvate carboxykinase,

and the phosphoenolpyruvate converted to glucose by a reversal of the reactions of the Embden-Meyerhof-Parnas pathway. Thus four molecules of acetyl-CoA will give rise to two molecules of succinate and this in turn will result in the

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formation of one molecule of glucose and the loss of two molecules of carbon dioxide (Fig. 6.5).

Figure 6.5
The pathway of incorporation of [¹⁴ C] from [1-¹⁴ C]-acetate (O) or
[2-¹⁴ C]-acetate (

) into glucose during gluconeogenesis in plant tissue.

The glyoxyllate cycle was first demonstrated in the bacterium Pseudomonas (Kornberg & Krebs, 1957) grown on two-carbon compounds, and has since been shown to operate in many micro-organisms and plant tissues. The evidence for the cycle is based on the presence of the two key enzymes, malate synthetase and isocitrate lyase, and on the distribution of ¹⁴ C in organic acids, sugars and carbon dioxide when the tissue is supplied with specifically labelled [¹⁴ C] acetate. Malate synthetase and isocitrate lyase are found in a wide variety of plant tissues (Carpenter & Beevers, 1958), particularly in fatty seedlings where they increase in activity during germination. Similarly the activities of aconitase and citrate synthetase increase in these tissues during germination. Incubation of castor bean endosperm tissue with [I-¹⁴ C] acetate or [2–¹⁴ C] acetate results initially in the formation of [¹⁴ C] malate, and subsequently the radioactivity from [I-¹⁴ C] acetate results in about an equal labelling of CO₂ and sucrose. In the cotyledons of germinating peanut and sunflower seedlings (Bradbeer & Stumpf, 1959) and castor bean endosperm (Canvin & Beevers, 1961), [1-¹⁴ C] acetate was converted to carboxyl-labelled malate and to sucrose in which the glucose moiety was labeled in the 3 and 4 carbons, while [2-¹⁴ ]C acetate gave rise to malate labelled in the methylene carbons and to sucrose where the glucose moiety was labelled in the 1, 2, 5 and 6 carbons. These patterns or labelling of the products of acetate metabolism are consistent with the operation of a glyoxyllate cycle in these tissues (Fig. 6.5).