Tema 7: Homofermentative Pathway

Chapter 14 Pages 383 - 402

Formation of acetyl-CoA from pyruvate

pyruvate

Acetyl-CoA + CO2 + NADH2

Pyruvatedehydrogenase

Acetyl-CoA + CO2 + H2Acetyl-CoA + formic acid

PyruvateFormate lyase

PyruvateFerredoxin

oxidoreductase

Anaerobically

Pyruvatedehydrogenase

1) Catalyze an oxidative decarboxylation.

2) It is found in aerobically grown Bacteria, mitocondria, but not in Archaea.

3) The product acetyl-CoA usually goes to the TCA cycle instead of to acetyl-P

HOOC-C-CH3 CH3CO-SCoA + CO2

O O

NAD NADH2

TCA cycle

HSCoA

PyruvateFormate lyase

1) Catalyze an oxidative decarboxylation.where the electrons remain in the carbonyl group.

2) The product acetyl-CoA usually goes to acetyl-P.

HOOC-C-CH3 + CoASH CH3CO-SCoA + CH2O2

O O

CH3CO-P + HSCoA

O

CH3COOH + ATP

Pi Phosphotransacetylase

Acetate kinase

ADP

Mg+

PyruvateFerredoxin

oxidoreductase

1) Catalyze an oxidative decarboxylation where ferredoxinis the Electron acceptor.

2) It is found typically in clostridia and sulfate reducing bacteria (SRB) and other anaerobes.

3) The product acetyl-CoA usually goes to acetyl-P.

HOOC-C-CH3 + CoASH CH3CO-SCoA + CO2

O O

Fd ox Fd red

2H+ 2H2

hydrogenase

CH3COOH + ATPAcetate kinase

ADPMg+

CH3CO-P + HSCoA

O

Pi

Phosphotransacetylase

How is acetyl-CoA made from Acetate?

It is typically made as follows

CH3COOH + ATPAcetate kinase

ADPMg+

CH3CO-P + HSCoA

O

Pi

Phosphotransacetylase

CH3CO-SCoA

O

acetylCoAsynthetase

or

INOUT

Carbon and energy

Lactic Acid Bacteria

Characteristics: Gram positive, carbohydrate users, proteolysis rare, nonmotile, non-spore forming

Strict fermentors, unable to synthesize cytochromes unless heme is

added. catalase negative oxidase negative

Nutritionally fastidious All make lactic acid (lactate) as predominant end

product

Lactic Acid Bacteria Types of fermentation Homofermentative: glucose to 2 lactic acids,

85-95% of glucose carbon in lactate Heterofermentative: glucose to 1 lactate, 1

ethanol, and 1 carbon dioxide, only 50% or less of glucose carbon in lactate.

Types of products will define the pathway used and ATP made.

Lactic Acid Bacteria

Types of organisms Streptococcus: homofermentative

Leuconostoc: heterofermentative

Pediococcus homofermentative

Lactobacillus; heterofermentative or homofermentative.

Lactic Acid Bacteria Streptococcus species: Enterococcus: gut dwellers Lactococcus

natural fermentations Lactic acid production: lowers pH,

preserves and precipitates proteins

Lactic acid bacteria Homofermentative pathway Uses Glycolytic pathway to make 2 pyruvates

from glucose Overview:

Activation-use 2 ATP Make ß-carbonyl C-C bond cleavage Oxidation/reduction Substrate-level phosphorylation

Glucose

Glucose-6-P

Homofermentative Pathway in Streptococci

ADPATP

Used 2 ATPMade 4 ATPNet ATP yield=2 ATP/glucose

Fructose-6-P

ADP

ATP

Fructose-1,6 bis P

Dihydroxyacetone-P 2 Glyceraldehyde-3-P

2 1,3-bisphosphoglycerate

2 3-phophoglycerate

2 2-phosphoglycerate

2 phosphoenolpyruvate

2 pyruvate

2 ADP

2 ATP

2 ADP2 ATP

2 NAD+

2 NADH

2 H2O

Dihydroxyacetone-Pconverted to glyceraldehyde-3-PPathway shows 2 G-3-P's afterthis step.

Reoxidation of NADH

2 pyruvate2 NADH

2 NAD+2 lactate

Lactate dehydrogenase

ATP

ADP

CH2

OH

OH OH OH

H HH

H

H

OC C C C CHO

Glycolytic Pathway for Glucose Metabolism

Glucose

Hexokinase or PTS systemATP

ADP

+

Glucose-6-P

Fructose-6-P

Fructose-1,6-bisP

Glyceraldehyde-3-P Dihydroxyacetone-P

G6P isomerase

Phosphofructo-kinase

Fructose-1,6-bis Paldolase

Triose isomeraseNow have 2 G3P's

CH2

OH

OH OH OH

H HH

H

H

OC C C C C=3OP O

=3OP O

H

CH2

OH

OH OH

H H

H O

C C C C CH2OH

=3OP O

H

CH2

OH

OH OH

H H

H O

C C C C CH2 PO3=

CH2

OH O

H H

C C=3OP O

OH

H O

C C CH2 PO3=H O

ATP

ADP

Glycolytic Pathway for Glucose Metabolism

Glyceraldehyde-3-P(Metabolism of only one G3P is shown)

=3OP O CH2

OH O

H H

C C

CH2

OH

OH

C C S-Enz=3OP O

CH2

OH

OH

C C=3OP O O-PO3

=

CH2

OH

OH

C C=3OP O OH

O-PO3=

HO-CH2

OH

C C OH

O-PO3=

CH2

O

C C OH

O

CH3

O

C C OH

{ }

NAD+

NADH

PO4=

H2O

ADP

ATP

1,3-bisphospho-glycerate

3-phosphoglycerate

2-phosphoglycerate

phosphoenolpyruvate

pyruvate

Triosephosphatedehydrogenase

Phosphoglyceratekinase

Phosphoglyceratemutase

Enolase

Pyruvatekinase

From the 3 and 4 carbonsof glucoseFrom the 1 and 6 carbons

of glucose

Mechanism of the isomeration reaction

H2CO PO3=

C

HCOH

HOCH

HCOH

H OH

OHC

O

H2CO PO3=

C

HCOH

HOCH

HCOH

H

H

C OH

H2CO PO3=

C

HCOH

HOCH

HCOH

O

HC OH

H

Glucose-6-P cis-enolateFructose-6-P

Isomerization Reaction:Creates an electron attracting keto group at the # 2 carbon

• H dissociates from C2• 2 electrons shift to form cis enediol• H from hydroxyl group dissociates• 2 electrons shift to form keto group.• Forces electrons in enol bond to shift to C1.

C-C bond cleavage: Aldolase Reaction

H dissociates from C4; 2 electrons shift to form cis enediolH from hydroxyl group (C4) dissociates2 electrons shift to form keto group.Forces electrons in enol bond to shift to C1.

H2CO PO3=

C O

H2CO PO3=

HCOH

HOCH

HC HO

CHOH

C O-

H2CO PO3=

H2CO PO3=

HCOH

CH O

+

C O

H2CO PO3=

C

H

OH

Mechanism of the aldolase reaction

Carbonyl betato Carbon with O

Enol formation

Dihydroxyacetone-P

Glyceraldehyde-3-P

Coenzymes (cofactors)/Vitamins Some are bound to enzyme

Apoenzyme + cofactor give holoenzyme Metal ion, organic cofactors

Some are soluble Act as co-substrate Pyruvate + NADH + H+ --> lactate + NAD+

Vitamins: Portion of cofactor that cell can’t make, must be in

diet “Vital amine”

N

C NH2

O

N

COOH

Vitamin Forms

Nicotinic acid(niacin) Nicotinamide

Vitamin forms: Niacin

Nutritional disease: pellagra

PO4

=in NADP+

NH2

N

C

O

NH2

O

OHOH

HH

H

CH2

H

O

OP

P

O

OHOH

HH

H

CH2O

O

HO

HO

O

N

NN

N

+

R

N

CC

CCC

+H

H

H

H

O

NH2C

H

H

R

N

CC

CCC

H H

H

O

NH2C

-2H

+2H

Oxidized Form Reduced Form

Nicotinamide Adenine Dinucleotide

NAD functions Function: oxidation reduction reaction,

accepts hydride anion (H-): one proton and two electrons

That’s why we write NADH + H +

Biosynthesis uses NADP+ most often Catabolism uses NAD+ most often.

In conclusion Streptococcus Uses glycolysis to degrade glucose to 2

pyruvates NADH’s made in pathway are reoxidized by

reducing pyruvate to lactate NADH is key cofactor in oxidation reduction

reactions ATP made solely by substrate level

phosphorylation.