Lecture 22

– New HW assignment– Anaerobic metabolism (continued)– Other sugars– Gluconeogenesis– Regulation

Alcoholic fermentation (yeast don't have Lactate DH)

2. alchohol dehydrogenase

C-O-

C=O

CH3

O

Pyruvate

H-C-O-H

H

NADH, H+

NAD+

1. Pyruvate decarboxylase (TPP) Mg2+

, thiamine pyrophosphate

CO2 CH3

H-C=OAcetaldehyde

Ethanol

CH3

Figure 17-26 Thiamine pyrophosphate (TPP).

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Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.

Mechanism of Pyruvate Decarboxylase using TPP

1. Nucleophilic attack by the dipolar cation (ylid) form of TPP on the carbonyl carbon of pyruvate to form a covalent adduct.

2. Loss of carbon dioxide to generate the carbanion adduct in which the thiazolium ring of TPP acts as an electron sink.

3. Protonation of the carbanion

4. Elimination of the TPP ylid to form acetaldehyde and regenerate the active enzyme.

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Figure 17-25 The two reactions of alcoholic

fermentation.

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Figure 17-30The reaction mechanism of alcohol dehydrogenase involves direct hydride transfer of the

pro-R hydrogen of NADH to the re face of acetaldehyde.

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Alcoholic fermentation

2ADP + 2 Pi

2 ATP

Glucose 2 Ethanol + 2 CO2

Pyruvate decarboxylase is present in brewer's yeast but absent in muscle / lactic acid bacteria

Other types of fermentations also exist…

CoASH

pyruvate acetyl-CoA + acetyl-P acetate

acetaldehyde

ethanol

Mixed acid: (2 lactate + acetate + ethanol)so, in addition to lactate production…

NADH, H+

NAD+

ADP ATP

NADH, H+

NAD+

lactate

Butanediol fermentation

C-O-

C=O

CH3

O

2 Pyruvate

NADH, H+

NAD+

CO2

Acetolactic acid

C-O-

C-C-O-CH3

H

O

O

CH3

CO2

C=O

CH3

CH3

HC-OH

Acetoin

CH3

CH3

HC-OH

HC-OH

2,3-butanediol

Other fermentations (Clostridium)

CoAH2

CH3-C-COOH

CO2

O

CH3-C-CoA

O

Acetyl-CoA

CoA

CH3-C-CH2-C-CoA

O O

CoA

Acetic acid

CO2

CoA

CH3-C-CH3

O

acetone

CH3-C-CH3

OH

isopropanol

NADH

Other fermentations (Clostridium)

H2O

NADHNAD

CH3-C-CH2-C-CoA

O O

CH3-CH=CH-C-CoA

O

CH3-CH2CH2-C-CoA

O

CH3-CH2CH2-C-OH

O H2O

2 NADH 2 NAD

CH3-CH2CH2-CH2-OHbutanol

butyric acid

What about other sugars?

Fructose - fruits, table sugar (sucrose).

Galactose - hydrolysis of lactose (milk sugar)

Mannose - from the digestion of polysaccharides and glycoproteins.

All converted to glycolytic intermediates.

Fructose metabolism

Two pathways: muscle and liverIn muscle, hexokinase also phosphorylates fructose producing F6P.

Liver uses glucokinase (low levels of hexokinase) to phosphorylate glucose, so for fructose it uses a different enzyme set

Fructokinase catalyzes the phosphorylation of fructose by ATP at C1 to form fructose-1-phosphate.

Type B aldolase (fructose-1-phosphate aldolase) found in liver cleaves F1P to DHAP and glyceraldehyde.

Glyceraldehyde kinase converts glyceraldehyde to GAP.

Fructose metabolism

Glyceraldehyde can also be converted to glycerol by alcohol dehydrogenase.

Glycerol is phosphorylated by glycerol kinase to form glycerol-3-phosphate.

Glycerol-3-phosphate is oxidized to DHAP by glycerol phosphate dehydrogenase.

DHAP is converted to GAP by TIM

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Figure 8.16c Important disaccharides formed by linking monosaccharides with O-glycosidic bonds.

Lactose, milk sugar.

Galactose metabolism

Galactose is half the sugar in lactose.

Galactose and glucose are epimers (differ at C4)

Involves epimerization reaction after the conversion of galactose to the uridine diphosphate (UDP) derivative.

1. Galactose is phosphorylated at C1 by ATP (galactokinase)

2. Galactose-1-phosphate uridylyltransferase transfers UDP-glucose’s uridylyl group to galactose-1-phosphate to make glucose-1-phosphate (G1P) and UDP-galactose.

3. UDP-galactose-4-epimerase converts UDP-galactose back to UDP glucose.

4. G1P is converted to G6P by phosphoglucomutase.

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Mannose metabolism

Mannose is found in glycoproteinsEpimer of glucose at the C2 positionConverted to F6P by two-step pathway

1. Hexokinase converts mannose to mannose-6-phosphate

2. Phosphomannose isomerase converts the aldose to ketose F6P. (the mechanism is similar to phosphoglucose isomerase with an enediolate intermediate).

Figure 17-37 Metabolism of mannose.

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Entner-Doudoroff pathway

Although glycolysis is nearly universal, some bacteria use an alternate route called the Entner-Doudoroff pathway.

Final product is ethanol.

Figure 17-38Entner–Doudoroff pathway for glucose breakdown.

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Sugar catabolic pathwaysGlycolysisLactate fermentationAlcohol fermentationFructose metabolismGalactose metabolismMannose metabolismEntner-Doudoroff pathway

GluconeogenesisGluconeogenesis-production of glucose under starvation

conditions since some cells (brain and red blood cells) can only use glucose as a carbon source.

Noncarbohydrate precursors (lactate, pyruvate, citric acid cycle intermediates, and carbon skeletons of most amino acids) can be converted to glucose.

Must go through oxaloacetate (OAA) first.Lysine and leucine cannot be converted to glucose (degrade to

acetyl-CoA)Fatty acids cannot be converted to glucose precursors in

animals-degraded completely to acetyl-CoAPlants can convert fatty acids to glucose with the glyoxylate

cycle.Glycerol can be converted to to glucose via a DHAP

intermediate

•3 steps (1, 3, 10) are considered irreversible due to energetics and inhibitors preventing the back reaction.•Purpose of gluconeogenesis is to supply free glucose for use by brain or storage during energy excess.•Generally done in the liver.

-4 kcal

-3.4 kcal

+5.7 kcal

+0.4 kcal

+1.5 kcal

-4.5 kcal

+1.06 kcal

+0.4 kcal

-7.5 kcal

Gluconeogenesis (new glucose formation)

• Mainly occurs in the liver. • Shares 7 reversible steps with glycolysis-but must have a mechanism

around the irreversible steps (all Gº’ must be negative).Step 1

PEP Pyruvate Gº’= -7.5

ADP ATP

Pyruvate kinase

Overcome by circuitous route…

PEPPyruvate

Gº’= +0.2CO2

ATPbiotin

ADP

Pyruvate carboxylase

GTP GDP

PEP carboxykinase

OAA

CO2

Pyruvate is converted to OAA before PEP

Pyruvate carboxylase catalyzes the ATP driven formation of oxaloacetate from pyruvate and bicarbonate.

PEP carboxykinase (PEPCK) converts oxaloacetate to PEP in a reaction that uses GTP as a phosphorylating agent.

Pyruvate carboxylaseP

age

602

Has a biotin prosthetic group

Biotin enzymes often used for carboxylations with bicarbonate by forming a carboxyl substituent at its ureido group.

Biotin is an essential human nutrient.

Binds tightly to avidin and streptavidin (can be used as a linking agent in biotechnological applications b/c of high affinity).

Figure 23-3aBiotin and carboxybiotinyl–enzyme. (a) Biotin consists of an imidazoline ring that is cis-fused to a tetrahydrothiophene ring bearing a valerate side chain.

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Figure 23-3bBiotin and carboxybiotinyl–enzyme. (b) In carboxybiotinyl–enzyme, N1 of the biotin ureido group is

the carboxylation site.

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Long flexible chain

Enzyme

Figure 23-4 Two-phase reaction mechanism of pyruvate carboxylase.

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Figure 23-4 (continued) Two-phase reaction mechanism of pyruvate carboxylase. Phase II

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Pyruvate carboxylase

Regulated by acetyl-CoA. (allosteric activator)

Inactive without bound acetyl-CoA.

Inhibition of the citric acid cycle by high levels of ATP and NADH causes oxaloacetate to undergo gluconeogenesis.

PEPPyruvate

Gº’= +0.2CO2

ATPbiotin

ADP

Pyruvate carboxylase

GTP GDP

PEP carboxykinase

OAA

CO2

PEP CarboxykinaseP

age

602

Monomeric 608 aa enzyme.

Catalyzes the GTP driven decarboxylation of OAA to PEP forming GDP

PEPCK cellular location varies with species

In mouse and rat liver it is in the cytosol

In pigeon and rabbit liver it is mitochondrial

In humans and guinea pigs it is in both.

Figure 23-5 The PEPCK mechanism.P

age

847

OAA

Gluconeogenesis requires transport between the mitochondria and cytosol

Enzymes for converting PEP to glucose are in the cytosol.

Intermediates need to cross barriers in order for gluconeogenesis.

OAA must leave the mitochondria for conversion to PEP or PEP formed in the mitochondria must go to the cytosol.

PEP tranported across the membrane by specific proteins.

Oxaloacetate has no specific transport system.

OAA must be convertted to either aspartate or malate

Gluconeogenesis requires transport between the mitochondria and cytosol

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The difference between the 2 routes for OAA involves the transport of NADH.

Malate dehydrogenase requires reducing equivalents to travel from the mitochondria to the cytosol. (uses mitochonridal NADH and produces cytosolic NADH).

Aspartate aminotransferase does not use NADH.

Cytosolic NADH required for gluconeogenesis so usually goes through malate.

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Hydrolytic reactions bypass PFK and Hexokinase

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Instead of generating ATP by reversing the glycolytic reactions, FBP and G6P are hydrolyzed to release Pi in an exergonic reaction.

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Gluconeogenesis

Glucose + 2ADP + 2Pi + 2NAD+ 2 Pyruvate + 2ATP + 2NADH + 4H+ + 2H2O

Net reaction

2ADP + 2GDP + 4Pi2ATP + 2GTP + 4H2O

Glycolysis

2 Pyruvate + 4ATP + 2GTP 2NADH + 4H+ + 6H2O

Glucose + 4ADP +2GDP + 6Pi + 2NAD+

Control Points in Glycolysis

1st reaction of glycolysis (Gº’ = -4 kcal/mol)

OH1

OHO

OH

HOOH

*2

3

4

56

Glucose

OH1

O

-2O3P-O

OH

HOOH

*2

3

4

56

ATP

ADP

Glucose-6-phosphate (G6P)

Hexokinase (HK) I, II, IIMuscle(II), Brain (I)

Mg2+

Mg2+

Glucokinase (HK IV) in liver

Regulation of Hexokinase

• Glucose-6-phosphate is an allosteric inhibitor of hexokinase.

• Levels of glucose-6-phosphate increase when downstream steps are inhibited.

• This coordinates the regulation of hexokinase with other regulatory enzymes in glycolysis.

• Hexokinase is not necessarily the first regulatory step inhibited.

Types of regulation

1. Availability of substrate Glucokinase (KM 12 mM) vs. HK (KM = 0.01 - 0.03 mM)

2. Compartmentalization -Brain vs. Liver vs. Muscle (type I mitochondrial membrane, type II cytoplasmic)

3. Allosteric regulation - feedback inhibition by G-6-P, overcome by Pi in type I (Brain/ mitochondrial controlled by Pi levels)

4. Hormonal regulation. Liver has HK as fetal tissue. Changes to glucokinase after about 2 weeks. If there is no dietary carbohydrate, no glucokinase. Must have both insulin and carbohydrates to induce.

2 places where there is no net reaction

1. ATP + F-6-P F-1,6-P2 + ADP

2. F-1,6-P2 F-6-P + Pi

PFK

F-phosphataseMg2+

Mg2+

Net: ATP ADP + Pi + heat

Similar reaction occurs with hexokinase and G-6-phosphatase.Generally regulated so this does not occur (futile cycle).

May function in hibernating animals to generate heat.

Primary regulation - reciprocal with energy charge

Enzyme + -

Hexokinase G-6-P

PFK Pi, ADP, AMP, F-6-P,

F-2,6-P2

ATP, citrate, NADH

F-6-phosphatase

ATP AMP, F-2,6-P2

Pyruvate kinase

K+, AMP, F-2,6-P2

ATP, acetyl-CoA, cAMP

Pyruvate carboxylase

Acetyl-CoA

Major regulation is through energy charge

ATP

ATP

Gluconeogenesis

GlycolysisADP

Same reactions make AMP or ADP (primarily in lipid and nucleotide metabolism)

AMP + ATP 2 ADPAdenylate kinase

[ATP] +1/2[ADP]

[AMP] + [ADP] + [ATP]Energy charge

1.0 = 100% ATP Body generally likes it close to 0.90.5 = 100% ADP0 = 100% AMP

Regulation of PhosphoFructokinase (PFK-1)

• PKF-1 has quaternary structure• Inhibited by ATP and Citrate• Activated by AMP and Fructose-2,6-

bisphosphate• Regulation related to energy status of cell.

PFK-1 regulation by adenosine nucleotides

• ATP is substrate and inhibitor. Binds to active site and allosteric site on PFK. Binding of ATP to allosteric site increase Km for ATP

• AMP and ADP are allosteric activators of PFK. • AMP relieves inhibition by ATP.

• ADP decreases Km for ATP

• Glucagon (a pancreatic hormone) produced in response to low blood glucose triggers cAMP signaling pathway that ultimately results in decreased glycolysis.

Effect of ATP on PFK-1 Activity

Effect of ADP and AMP on PFK-1 Activity

Regulation of PFK by Fructose-2,6-bisphosphate

• Fructose-2,6-bisphosphate is an allosteric activator of PFK in eukaryotes, but not prokaryotes

•Formed from fructose-6-phosphate by PFK-2

•Degraded to fructose-6-phosphate by fructose 2,6-bisphosphatase.

•In mammals the 2 activities are on the same enzyme

•PFK-2 inhibited by Pi and stimulated by citrate