Molecular Biochemistry I

Gluconeogenesis;
Regulation of Glycolysis &Gluconeogenesis

Contents of this page:
Reactions of gluconeogenesis
Summary of gluconeogenesis pathway
Reciprocal regulation of gluconeogenesis & glycolysis
Cori cycle

Gluconeogenesis occurs mainly in liver. Gluconeogenesis occurs to a more limited extent in the kidney and small intestine under some conditions. 

Synthesis of glucose from pyruvate utilizes many of the same enzymes as Glycolysis. Three reactions of Glycolysis have such a large negative DG in the forward direction that they are essentially irreversible (see lecture notes on Glycolysis):
   
Hexokinase
(or Glucokinase), Phosphofructokinase, and Pyruvate Kinase.
These steps must be bypassed in Gluconeogenesis. Two of the bypass reactions involve simple hydrolysis reactions.

Below is the forward reaction catalyzed by each of these Glycolysis enzymes, followed by the bypass reaction catalyzed by the Gluconeogenesis enzyme.

Hexokinase or Glucokinase (Glycolysis) catalyzes:
   glucose + ATP glucose-6-phosphate + ADP

Glucose-6-phosphatase (Gluconeogenesis) catalyzes:
   glucose-6-phosphate + H2O glucose + Pi

Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells. Evidence indicates that the catalytic site is exposed to the ER lumen. Another subunit of the enzyme is postulated to function as a translocase, providing access of substrate to the active site.

 

Phosphofructokinase (Glycolysis) catalyzes:
   fructose-6-phosphate + ATP fructose-1,6-bisphosphate + ADP

Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes:
  
fructose-1,6-bisphosphate + H2O fructose-6-phosphate + Pi


Pyruvate Kinase
(last step of Glycolysis) catalyzes:
  
phosphoenolpyruvate + ADP pyruvate + ATP

For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative DG of phosphate hydrolysis than ATP.

The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:

(a) Pyruvate Carboxylase (Gluconeogenesis) catalyzes:
    pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi

(b) PEP Carboxykinase (Gluconeogenesis) catalyzes:
    oxaloacetate + GTP phosphoenolpyruvate + GDP + CO2

Contributing to spontaneity of the two-step pathway are the following:
  • Free energy of cleavage of one ~P bond of ATP is conserved in the carboxylation reaction. Spontaneous decarboxylation contributes to spontaneity of the 2nd reaction (PEP synthesis).
  • Cleavage of a second ~P bond of GTP also contributes to driving synthesis of PEP.

Pyruvate Carboxylase utilizes biotin as prosthetic group. See diagram p. 846 of Biochemistry, 3rd Edition, by Voet & Voet.

Biotin has a 5-carbon side chain whose terminal carboxyl is in an amide linkage to the e-amino group of a lysine of the enzyme.

The biotin and lysine side chains together form a long swinging arm that allows the functional group of biotin to swing back and forth between two active sites.

Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase.

ATP reacts with HCO3- to yield carboxyphosphate. The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring system. Overall:

biotin + ATP + HCO3- carboxybiotin + ADP + Pi

At the other active site of Pyruvate Carboxylase, the activated CO2 is transferred from biotin to pyruvate, as summarized below and at right:

carboxybiotin + pyruvate biotin + oxaloacetate

View at right an animation of the reaction sequence catalyzed by Pyruvate Carboxylase.


of Pyruvate Carboxylase 

The protein visualization exercise at right focuses on the biotinyl domain of another carboxylase enzyme, Acetyl-Coenzyme A Carboxylase. Explore the structure of biotin and its attachment via a lysine side chain. 


Biotinyl Domain 
of a Carboxylase

Pyruvate Carboxylase, which converts pyruvate to oxaloacetate, is allosterically activated by acetyl coenzyme A.  The adaptive value of this regulation relates to the interconnectness of the pathways shown at right.

Acetyl CoA enters Krebs Cycle by condensing with oxaloacetate, whose concentration tends to be limiting for Krebs Cycle. 

When Gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose (via PEP). Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The resulting increase in [acetyl CoA] activates Pyruvate Carboxylase to synthesize more oxaloacetate.

Avidin, a protein in egg whites with a b barrel structure, tightly binds biotin. Excess consumption of raw eggs can cause nutritional deficiency of biotin.

The strong avidin-to-biotin affinity is utilized by biochemists as a highly specific "glue." For example, if it is desired to bind 2 proteins together at some stage of an experiment, biotin may be covalently linked to one protein and avidin to the other.


Avidin-Biotin Complex

PEP Carboxykinase catalyzes GTP-dependent formation of phosphoenolpyruvate from oxaloacetate. See diagram p. 847.

The reaction is thought to proceed in two steps:
Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate. Phosphate transfer from GTP then yields phosphoenolpyruvate (PEP).

 

In bacterial PEP Carboxykinases, ATP is phosphate donor instead of GTP. In the crystal structure of an E. Coli PEP Carboxykinase at right, pyruvate is at the active site as an analog of phosphoenolpyruvate or oxaloacetate.

A metal ion such as Mn++ is required for the PEP Carboxykinase reaction, in addition to a Mg++ ion that binds with the nucleotide substrate at the active site. The Mn++ is thought to promote the phosphate transfer by interacting simultaneously with the enolate oxygen atom and an oxygen atom of the terminal phosphate of GTP or ATP.

Explore at right the structure of E. Coli PEP Carboxykinase. Structure solved by L. W. Tari, A. Matte, H. Goldie & L. T. J. Delbaere in 1997; PDB 1AQ2.

Recommended display options:

Select ligand and display as spacefill.
Identify and note relative positions of ATP, pyruvate, Mg++ and Mn++.
Note that the terminal phosphate of ATP would be transferred to the oxaloacetate substrate to yield phosphoenolpyruvate (pyruvate substituting here).

Select protein, display as cartoon with color structure.
Describe the unique secondary structure of this enzyme.

Now change the display of protein to spacefill, with color chain.
Speculate on whether a change in conformation of the enzyme might accompany binding of any of the substrates.


     C  O  N  P  Mg  Mn    

Gluconeogenesis inputs:  
The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism. Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these (see diagram p. 844, and web page on amino acid catabolism). Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. 
Glycerol
, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis.

Gluconeogenesis pathway is summarized below, with gluconeogenesis enzyme names in red and names of reversible glycolysis enzymes in blue:

Glycolysis & Gluconeogenesis pathways are both spontaneous. If both pathways were simultaneously active within a cell it would constitute a "futile cycle" that would waste energy. Overall, each pathway may be summarized as follows (ignoring water & protons):

Glycolysis:
glucose + 2 NAD+ + 2 ADP + 2 Pi
2 pyruvate + 2 NADH + 2 ATP

Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP
glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi

Glycolysis yields 2 ~P bonds of ATP.
Gluconeogenesis expends 6 ~P bonds of ATP and GTP.
A futile cycle consisting of both pathways would waste 4 ~P bonds per cycle.

To prevent this waste, Glycolysis and Gluconeogenesis pathways are reciprocally regulated

Local Control includes reciprocal allosteric regulation by adenine nucleotides.

This insures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP. When ATP is high it is more useful to the cell to store glucose as glycogen.
When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing glucose.

Global Control in liver cells includes reciprocal effects of a cyclic AMP cascade, triggered by the hormone glucagon when blood glucose is low. Phosphorylation of enzymes and regulatory proteins in liver by Protein Kinase A (cAMP-Dependent Protein Kinase) results in inhibition of glycolysis and stimulation of gluconeogenesis, making glucose available for release to the blood.

Proteins relevant to these pathways that are phosphorylated by Protein Kinase A include:

Reciprocal regulation by fructose-2,6-bisphosphate:

Recall that Phosphofructokinase, the rate-limiting step of the Glycolysis pathway, is allosterically inhibited by ATP. At high concentration, ATP binds to a low affinity regulatory site, promoting the tense conformation. Sigmoidal dependence of reaction rate on [fructose-6-phosphate] is observed at high ATP, as depicted at right. 

Phosphofructokinase activity in the presence of the globally controlled allosteric regulator fructose-2,6-bisphosphate is similar to that observed when [ATP] is low. Fructose-2,6-bisphosphate promotes the relaxed state, activating Phosphofructokinase even at relatively high [ATP].

Thus activation by fructose-2,6-bisphosphate, whose concentration fluctuates in response to external hormonal signals, supersedes local control by ATP concentration.

In the Biochemistry Simulations tutorial at right, select the module on Phosphofructokinase, and explore effects of varied concentrations of ATP and the activator fructose-2,6-bisphosphate on the dependence of reaction rate on fructose-6-phosphate concentration.   


Note: Hold down the Control key while clicking on the above icon.
 

The allosteric regulator fructose-2,6-bisphosphate is synthesized and degraded by a bi-functional enzyme that includes two catalytic domains: 
  • Phosphofructokinase-2 (PFK2) domain catalyzes:
    fructose-6-phosphate + ATP fructose-2,6-bisphosphate + ADP.

  • Fructose-Biosphosphatase-2 (FBPase2) domain catalyzes:
    fructose-2,6-bisphosphate + H2O fructose-6-phosphate + Pi

The bi-functional PFK2/FBPase2 assembles into a homodimer. (The structure of the rat testis enzyme at right was solved by M. H. Yuen, H. Mizuguchi, Y. H. Lee, P. F. Cook, K. Uyeda & C. H. Hasemann in 1998.)

Adjacent to the PFK2 domain in each copy of the liver enzyme is a regulatory domain subject to phosphorylation by cAMP-dependent Protein Kinase. Which catalytic domains of the enzyme are active depends on whether the regulatory domains are phosphorylated, as summarized below right.  

cAMP-dependent phosphorylation of the bi-functional enzyme activates FBPase2 and inhibits PFK2.

[Fructose-2,6-bisphosphate] thus decreases in liver cells in response to a cAMP signal cascade, activated by glucagon when blood glucose is low. Downstream effects include:

  • Glycolysis slows because fructose-2,6-bisphosphate is not available to activate Phosphofructokinase.

  • Gluconeogenesis increases because of the decreased concentration of fructose-2,6-bisphosphate, which would otherwise inhibit the gluconeogenesis enzyme Fructose-1,6-bisphosphatase.

View at right an animation showing regulation of PFK2/FBPase2, leading to synthesis or breakdown of fructose-2,6-bisphosphate. 


Synthesis & degradation of fructose-2,6-bisphosphate

Summarizing effects described above and in the notes on glycogen metabolism, a glucagon-induced cAMP cascade has the following effects in liver tissue:
  • Gluconeogenesis is stimulated.
  • Glycolysis is inhibited.
  • Glycogen breakdown is stimulated.
  • Glycogen synthesis is inhibited.
  • Free glucose formed for release to the blood.

The Cori Cycle operates during exercise.

For a brief burst of ATP utilization, muscle cells utilize ~P stored as phosphocreatine. Once phosphocreatine is exhausted, ATP is provided mainly by Glycolysis, with the input coming from glycogen breakdown and from glucose uptake from the blood. (Aerobic fat metabolism, discussed elsewhere, is more significant during a lengthy period of exertion such as a marathon run.)

Lactate produced from pyruvate passes via the blood to the liver where it may be converted to glucose. The glucose may travel back to the muscle to fuel Glycolysis.

The Cori Cycle costs 6 ~P in liver for every 2 ~P made available in muscle.
The net cost is 4 ~P.
Although costly in terms of "high energy" bonds, the Cori Cycle allows the organism to accommodate to large fluctuations in energy needs of skeletal muscle between rest and exercise.

The equivalent of the Cori Cycle also operates during cancer. If blood vessel development does not keep pace with growth of a solid tumor, decreased oxygen concentration within the tumor leads to activation of signal processes that result in a shift to anaerobic metabolism. Energy dissipation by the Cori Cycle, which expends 6 ~P in liver for every 2 ~P produced via Glycolysis for utilization within the tumor, is thought to contribute to the weight loss that typically occurs in late-stage cancer even when food intake remains normal.

Copyright 1998-2007 by Joyce J. Diwan. All rights reserved.

Additional material on Gluconeogenesis:
Readings, Test Questions, & Tutorial

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