The Gluconeogenic Pathway

The glycolytic pathway can be used for the synthesis of glucose from simpler molecules through gluconeogenesis. Gluconeogenesis is the synthesis of glucose from nonsugar sources, especially amino acids and TCA cycle intermediates. Running glycolysis in the synthetic direction requires that there be a way to bypass the three free energy drops in the pathway, that is, the pyruvate kinase, phosphofructokinase, and hexokinase steps. The reactions could be run in reverse; however, that wouldn’t be very efficient. Because of the relationship between free energy and equilibrium, only a very small amount of, for example, phosphoenolpyruvate can be made by reversing the pyruvate kinase step. The processes that have very little free energy change associated with them operate near equilibrium. Therefore, an addition of enzymatic products, for example, glyceraldehyde‐3‐phosphate and dihydroxyacetone phosphate, would simply drive the aldolase reaction in the direction of fructose‐1,6‐ bisphosphate. That is, the new equilibrium would favor the synthesis of hexoses.

Bypassing the pyruvate kinase step

Bypassing the pyruvate kinase step requires oxaloacetate. The oxaloacetate can come from either of two sources. First, various reactions can build up TCA cycle intermediates, among them oxaloacetate. For example, aspartic acid has the same carbon skeleton as oxaloacetate and ammonia can be removed by several means to yield oxaloacetate: 



Alternatively, oxaloacetate can be made from pyruvate by the addition of CO 2 in the mitochondrial matrix (see Figure  1). This anapleurotic reaction is catalyzed by pyruvate carboxylase. The pyruvate carboxylase reaction consumes an ATP bond and CO 2. In eukaryotes, the oxaloacetate thus formed can then be shuttled out of the mitochondria by several pathways.



                      Figure 1

  

Once in the cytosol, the oxaloacetate is decarboxylated and phosphorylated by the enzyme pyruvate carboxykinase (see Figure  2). (The nomenclature is confusing; try to remember that pyruvate carboxylase only adds CO 2, whereas pyruvate carboxykinase removes CO 2 and adds phosphate. In the pyruvate carboxykinase reaction, the CO 2 added to make oxaloacetate is removed, so the only net reaction in the series is the addition of a phosphate to pyruvate to make phosphoenolpyruvate).





                 Figure 2

However, the two‐step reaction consumes two high‐energy phosphate bonds to do this task. A general rule is that biosynthetic pathways use several small, energetically favored steps to bypass a single highly unfavored one in the catabolic pathway.

Bypassing the phosphofructokinase and hexokinase steps

The second step that must be bypassed is the phosphofructokinase reaction. The strategy for doing this is simple: A phosphatase hydrolyzes the phosphate at position 1 of fructose bisphosphate, leaving the singly phosphorylated fructose‐6‐phosphate:




If fructose bisphosphate phosphatase and phosphofructokinase were to both operate at high rates in the cell, a great amount of ATP would be broken down to no effect. The two reactions would oppose each other:




The sum of these two reactions is simply:


 

Fructose bisphosphate phosphatase is regulated by the same allosteric effectors as is phosphofructokinase, except in the opposite manner. For example, phosphatase is activated by fructose‐2,6‐bisphosphate, whereas phosphofructokinase is inactivated by it. If there were no coordinate regulation of these steps, the net result would be the runaway consumption of ATP in a futile cycle. The regulatory mechanism doesn t completely shut down either reaction; rather, it ensures that there is a greater flow of carbon in one direction or the other. The small amount of ATP that is consumed by the futile cycle is the cost associated with the regulation.

The hexokinase step is bypassed in the same manner as is the phosphofructokinase reaction, by a phosphatase that is activated by high concentrations of glucose‐6‐phosphate (an example of substrate‐level control). Note that this is the opposite of the effect of glucose‐6‐phosphate on the rate of the hexokinase step.