Chapter 17

Gluconeogenesis

Why Gluconeogenesis? Gluconeogenesis is the synthesis of glucose from

noncarbohydrate precursors (pyruvate, amino acids, glycerol).

Gluconeogenesis is especially important during fasting or starvation, as glucose is the primary fuel for the brain and the only fuel for red blood cells (no mitochondria), and the reserve of glucose in body is used up in ~ 1 day without eating food. (During intense exercise, gluconeogenesis also takes place.)

The major site of gluconeogenesis is the liver. Small amount of gluconeogenesis can also occur in the kidney.

Gluconeogensis in liver and kidney maintain the glucose level in blood, from which it can be extracted by the brain and muscle to meet their needs.

The gluconeogenic pathway converts pyruvate into glucose. Other noncarbohydrate precursors, including lactate, amino acids

and glycerol, are converted to pyruvate or later intermediates.

A. Lactate (muscle-derived) can be converted to pyruvate in theliver (reverse of the lactic acid fermentation).

B. The carbon skeletons of most amino acids (almost all, only exceptions are lysine and leucine) can be converted into glucogenic intermediates (pyruvate, oxaloacetate) that enter gluconeogenesis.

C. Glycerol (end product of hydrolysis of triacylglycerols), can be converted into dihydroxyacetone phosphate (DHAP), which can enter either gluconeogenesis or glycolysis.

Most enzymes (7 enzymes) that can catalyze the reversible reactions are common in glycolysis and gluconeogenesis.

However, the three irreversible steps in glycolysis are too energetically favorable in one direction and must be bypassed in gluconeogenesis due to large free energy cost in the reverse direction.

Phosphofructose

kinase

phosphoglucose

isomerase

Aldolase

Glyceraldehyde 3-

phosphate dehydrogenase

Triose

phosphate

isomerase

Phosphoglycerate

kinase

Phosphoglycerate

mutase

Enolase

Stage 1 of Gluconeogenesis (7 reactions)

Two enzymes: Pyruvate carboxylase (in mitochondria) and PEP carboxykinase (in cytoplasm) are used to reverse the action of pyruvate kinase.

These occur at a cost of 1 ATP and 1 GTP per pyruvate that involves a carboxylation and a decarboxylation-coupled phosphorylation.

The other reactions in this stage use the same enzymes in glycolysis.

2X

Stage 2 of Gluconeogenesis (4 reactions)

Fructose 1,6-bisphosphatase (that catalyzes hydrolysis of a phosphoryl group) is used to reverse PFK.

Glucose-6-phosphatase is used to reverse hexokinase.

The hydrolysis of phosphorylgroup in these two steps are energetically favorable.

To form 1 glucose from 2 pyruvate requires total 6 NTPs.

The formation of phosphoenolpyruvate from pyruvate requires two enzymes and the reactions occur in mitochondria and cytoplasm, separately. The sum of the two reactions is:

Phosphoenolpyruvatecarboxykinase

mitochondria

cytoplasm

Pyruvate carboxylase requires the vitamin biotin (VB7) as a cofactor.

The formation of oxaloacetate (OA) by pyruvate carboxylase occurs in three steps (involves four substrates, sequential mechanism).

Step 1:

Biotin, a CO2 carrier

Pyruvate Carboxylase

Activation of bicarbonate by ATP (phosphorylation)

Carboxylated biotin-enzyme

Step 2 :

Step 3 :

Activated CO2 group is transferred to the biotin-enzyme

complex covalently.

Carboxylate group is transferred to the substrate pyruvate.

The long flexible linker between biotin and the enzyme (provided by 5C side chain on biotin and 4C lysine) enables the carboxy-biotin to rotate from one active site (ATP-biocarbonate site) to the other (pyruvate site) sequentially.

Swing Arm of the Biotin Coenzyme

Biotin

Acetyl CoA (entry point of citric acid cycle) allosterically activate the pyruvate carboxylase (entry point of gluconeogenesis, the product oxaloacetate is also in the entry point of citrate acid cycle). This important control mechanism balances the two substrates for citrate synthase.

Pyruvate

pyruvate carboxylase

citric acid cycle

Pyruvate to oxaloacetate conversion occurs in mitochondrion matrix.

In order to be translocated to the cytoplasm, oxaloacetate is reducedby NADH to malate by malate dehydrogenase.

Malate is able to be transported from mitochondrion matrix into the cytoplasm (by malate transporter).

Malate is then oxidized back to oxaloacetate by NAD+ and form cytoplasmic NADH.

Malate

dehydrogenase

Malate

dehydrogenase

Transport of oxaloacetate from mitochondria matrix to cytoplasm

Phosphoenolpyruvatecarboxykinase

Hydrolysis of GTP does not provide enough free energy to phosphorylate oxaloacetate. As a result, decarboxylation, which releases energy, is needed to drive the reaction forward (the reason for the carboxylation in the first step at the cost of an ATP)

Two enzymes (pyruvate carboxylase and phosphenolpyruvatecarboxykinase) catalyze the phosphorylation of pyruvate at the cost of two NTPs. The location of reaction also switch from mitochondrion matrix to cytoplasm though a malate transporter (redox reaction).

Decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate

Phosphoenolpyruvate (PEP) is metabolized by the same series of enzymes of glycolysis in the reverse direction in cytoplasm (enolase, phosphoglycerate mutase (PGM), phosphoglyceratekinase (PGK), GAPDH, TIM, aldolase, in condition that favors gluconeogenesis) until the next irreversible step: the hydrolysis of fructose 1,6-bisphosphate.

The enzyme catalyzing this reaction is fructose 1,6-bisphosphatase, an allosteric enzyme that participate in regulation.

G6P is Converted to Glucose in ER F6P->G6P isomerization is easy and reversible. In most tissues,

gluconeogenesis stops here and G6P is used to form glycogen.

The generation of free glucose occurs essentially only in liver, and is the final irreversible step in gluconeogenesis.

G6P is transported into the lumen of the endoplasmic reticulum.

Glucose 6-phosphatase, an integral membrane protein on the inner surface of the endoplasmic reticulum, catalyzes the formation of glucose from G6P.

Glucose is then transported to cytoplasm and to blood.

Hydrolysis of 4 more NTPs makes the gluconeogenesis reactions energetically favorable. (but costly)

Net reaction of gluconeogenesis:

Compare with direct reverse of glycolysis

2

4 ATP, 2 GTP expenditure

Gluconeogenesis

Gluconeogenesis and glycolysis are both energetically favorable pathways (both have overall large negative free energy changes). Therefore, there is no thermodynamic barrier to prevent them from taking place simultaneously.

The higher energy cost of gluconeogenesis (6 NTP vs. 2 ATP) means simultaneous activation of both pathways is not energy efficient.

Gluconeogenesis and glycolysis are regulated so that within a cell, one pathway is relatively inactive while the other is highly active. This is known as reciprocal regulation.

Glycolysis will predominate when glucose is abundant, and gluconeogenesis will be more active when glucose is scarce.

When ATP is required (AMP and ADP are high) and glucose is abundant (F-2,6-BP, F-1,6-BP are high), glycolysis predominates.

When the energy charge is high and biosynthetic intermediates are abundant (ATP, citrate, alanine, acetyl CoA are high), gluconeogenesis will take over.

The inter-conversion of F-1,6-BP and F-6-P by PFK or Fructose 1,6-bisphosphatase is a key regulatory site.

Inter-conversion of PEP and pyruvate is also regulated.

F-2,6-BP and AMP stimulates PFK and inhibits F-1,6-BPase. Citrate acts oppositely.PFK is also inhibited by ATP and low pH.

F-1,6-BP stimulates PK. ADP inhibits PC and PEPCK. ATP and alanine inhibit PK. Acetyl-CoA stimulate PC.

The key regulator molecule of glucose metabolism in liver is F-2,6-BP, which stimulates PFK and inhibits FBPase.

The kinase that synthesizes F-2,6-BP and the phosphatase that hydrolyzes it are located on the same polypeptide chain as two linked domains (PFK2/FBPase2), thus is called a bifunctional enzyme.

Bifunctional PFK2/FBPase2

Kinase: F6P -> F2,6BPPhosphatase: F2,6BP -> F6P

Regulation of PFK2/FBPase2 by GlucagonGlucagon, a peptide hormone, functions to raise glucose level, opposite to insulin. Together they maintain a relative constant blood glucose level. When blood glucose is low, glucagon level is high. Glucagon signaling pathway leads to phosphorylation of the bifunctional enzyme (by protein kinase A), which inhibits the kinase (no F-2,6BP, low PFK activity, slows down glycolysis), and stimulates the phosphatase (favors gluconeogenesis).When blood glucose is high, glucagon level is low. This leads to dephosphorylation of the bifunctional enzyme, that activate the kinase and produces more F-2,6BP, thus stimulating PFK to turn on glycolysis.

Substrate cycle: combinations of two pathways in which the final products from one is exactly the initial reactants of the other, and vice versa. Also referred to as futile cycles.

Despite reciprocal control, one pathway is never completely shut down. Why dont the body prevent futile cycles?

One hypothesis: substrate cycles can amplify a metabolic signal to increase the substrate flux down a metabolic pathway without changing the rates of opposing reactions too much.

Skeletal muscle (as well as red blood cell) and liver display inter-organ cooperation in a series of reactions called the Cori cycle.

Lactate produced by skeletal muscle and red blood cells is released into the blood.

Liver removes most of the lactate in blood and either converts it back into glucose (via gluconeogenesis), which can be released into the blood available to other tissues, or use the converted pyruvate for energy generation further down the citric acid cycle.

The Cori Cycle Converting Lactate back to Glucose

The Cori Cycle Converting Lactate back to Glucose

Both processes are active in the body but isolated in different organs.Blood transport the precursors and products around to where these are need. Membrane transporters allow them to be transported in and out of the cells.

Anaerobic metabolism

Alanine, like lactate, is a major precursor of glucose in liver.

Alanine is generated in muscle cells when the carbon skeleton of some amino acids are used to generate energy, the amino group goes to pyruvate to form alanine, which is released into blood.

Liver takes up the alanine and converts it back to pyruvate and then glucose that can be used by other tissues.

alanine pyruvatelactate

or Alanineor Alanine

Summary

1. Glucose can be synthesized from noncarbohydrate

precursors (pyruvate, lactate, amino acids, glycerol).

Gluconeogenesis requires four new reactions (enzymes) to

bypass the three irreversible reactions in glycolysis.

1. Gluconeogenesis and glycolysis are reciprocally regulated,

so both are not maximally active at the same time. Key

regulation points are the phosphofructokinase and fructose

1,6-bisphosphatase by F-2,6-BP. Pyruvate kinase and

pyruvate carboxylase are also regulated by other effectors

(ATP, ADP, AMP, alanine, F-1,6-BP, acetyl CoA) so they

are not active at the same time.

2. Precursors (lactate, alanine) produced by muscle are used

to synthesize glucose in liver that can be used by other

organs.

The following sequence is a part of the sequence of reactions in gluconeogenesis.

Match the capital letters representing the reaction in the gluconeogenic pathway

with parts a, b, c, etc.

(a) takes place in mitochondria.

(b) takes place in the cytoplasm.

(c) produces CO2.

(d) consumes CO2.

(e) requires NADH.

(f) produces NADH.

(g) requires ATP.

(h) requires GTP.

(i) requires thiamine.

(j) requires biotin.

(k) is regulated by acetyl CoA.

A, B

C, DD

AB

CA

D

A

A

none

Counting high-energy compounds:

How many NTP molecules are required to synthesize glucose

from each of the following compounds?

(a) Glucose 6-phosphate

(b) Fructose 1,6-bisphosphate

(c) Two molecules of oxaloacetate

(d) Two molecules of dihydroxyacetone phosphate

(e) Pyruvate

(f) Lactate

(g) 3-phosphoglycerate

none

none

2 ATP + 2 GTP

none

6 NTP

6 NTP

2 ATP

Match up. Indicate which of the conditions listed in the right-

hand column increase the activity of the glycolytic and

gluconeogenic pathways.

2

1

3

4 5

6

7

9

8

Gluconeogenesis takes place in liver during intense exercise,

which seems counterintuitive. Why would an organism

synthesize glucose and, at the same time, use glucose to

generate energy?

Compare the roles of lactate dehydrogenase in gluconeogenesis

and in lactic acid fermentation.