Chapter 16 Glycolysis and gluconeogenesis

§ Glycosis is an energy-conversion pathway in many organisms

§ The glycolytic pathway is tightly controlled

§ Glucose can be synthesized from noncarbohydrate precursors

§ Gluconeogenesis and glycolysis are reciprocally regulated

Glucose fates

Glucose: is an important fuel for most organisms

the only fuel that the brain uses under nonstarvation conditions

the only fuel that red blood cells can use at all

almost all organisms exist a similar process for glucose

p. 435 speculate the reasons

A key discovery was made by Hans Buchner and Eduard Buchner in 1897, quite by accident.

To manufacture cell-free extracts of yeast for possible therapeutic use,

replace phenol

Try sucrose (non-reducing sugar), sucrose was rapidly fermented into alcohol

by the yeast juice, sucrose fermentation

Fermentation could take place outside living cells

1860 Louis Pasteur: fermentation is inextricably tied to living cells.

Open the door to modern biochemistry

Lactate fermentation in muscle extracts

Glycosis is known as the Embden-Meyerhof pathway

Glucose is generated from dietary carbohydrates

is an important fuel for most organisms

Starch and glycogen: are digested by -amylase released by pancreas and saliva. The products

are maltose and maltotriose and the undigested product, limit dextrin.

Maltase, -glucosidase, -dextrinase

Sucrase, lactase

Synthesis high mannose type oligosaccharide to develop HIV-1 vaccine (Man4)

Chen CY, Wong CH (2007) Master thesis, NTU The side-effects of anti-reverse transcriptase

§ 16.1 Glycolysis – an energy-conversion pathway

three stages 1. consume energy

2. 6C is cleaved into 2 phosphorylated 3C

3. energy production

– takes place in the cytoplasm

invest

Stage 1 of glycolysis

*

*

*

**

**

Trap Glc

Aldose

6 ring

Ketose

5 ring

p. 438 bis- vs. di-

Hexokinase: requires Mg2+ or Mn2+

On Glc binding

Conformation markedly change

except the – OH of C6 is not

surrounded by protein,

phosphorylation

Other kinase to form a complex with ATP

12

*

*

*

*

p. 427 lyase

*

*

isomerase

Stage 2 of glycolysis

F1,6-bisP

***

*

major in equilibrium The subsequent reaction remove G3P

TPI or TIM

TPI structure:

8 parallel strands surrounded

by 8 helices

a general acid-base rx.

Glu 165, His 95

a kinetically perfect enzyme

kcat/KM: 2 108 M-1 s-1

close to the diffusion-controlled limit

p. 221-222

One international unit of enzyme:

the amount that catalyzes the formation of 1 mole of production in 1 min.

the conditions of assay must be specified.

Katal:

one katal is that amount of enzyme catalyzing the conversion of 1 mole of

substrate to product in 1 sec.

1 katal = 6 × 107 international units

His stabilize the negative charge that develops on the C-2 carbonyl group

methyl glyoxal + Pi

The active site is kept closed until the desired rx. takes place.

H of C1

H of C2

TPI suppresses an undesired side rx.

Stage 3 of glycolysis

A high phosphoryl-transfer potential

high-energy compound

preserve energy

Two processes must be coupled

Carboxylic acid compound

Cys149

His176

Hemithioacetal

p. 306

NAD+1

NAD+2NADH1 release

polarization

Aldehyde

acid

Energy released by carbon oxidation

High energy compound

p. 442

p. 420

Substrtate-level phosphorylation

Intracellular shift

Substrtate-level phosphorylation

*

**CO2*

*reversible

3 phosphoglycerate 2 phosphoglycerate

Enz-His-phosphate + 3 phosphoglycerate Enz-His + 2,3-bisphosphoglycerate

Enz-His + 2,3-bisphosphoglycerate Enz-His-phosphate + 2 phosphoglycerate

Glc + 2 Pi + 2 ADP + 2 NAD+

2 Pyr + 2 ATP + 2 NADH + 2 H+ + 2 H2O

The diverse of fates of pyruvate

Labeling isotope C3, C4

recycling

Fermentation:

An ATP-generating process in which organic compounds act as both donors and acceptors of electrons. Fermentation can take place in the absence of O2.

Pyruvate ethanolin yeast and several organisms

thiamine pyrophosphate zinc ion

Centrum

Glc + 2 Pi + 2 ADP + 2 H+ 2 ethanol + 2 ATP + 2 CO2 + 2 H2O

p. 446 (Fig. 16.10)

Pyruvate lactate occur in higher organisms, the amount of oxygen is limiting

Glc + 2 Pi + 2 ADP 2 Lactate + 2 ATP + 2 H2O

Magnesium lactate: a gel constituent; inhibit the production of

histamine by histidine decarboxylase

lactose

Obligate anaerobes:– organisms cannot survive in the presence of O2

Facultative anaerobes: organisms can function in the presence or absence of O2

CAM

via microorganisms

Watermelon juice: facilitate ethanol biofuel production

Biotech. for Biofuels (2009) 2: 18

NAD+ binding region in dehydrogenaseG3P dehydrogenase, alcohol dehydrogenase, lactate

dehydrogenase

Nicotinamide adenine dinucleotide

Rossmann fold

4 helices

6 parallel sheet

p. 449

Entry point in glycolysis of galactose and fructose

Fructose metabolism

(liver)

2ATP

F 6-P

hexokinase

(adipose tissue)

affinity

compartment

Galactose metabolism

hexokinase

Galactose metabolism

Polysaccharides Glycoproteins

G6P

mutase

p. 314

Lactose intolerance (hypolactasia) – a deficiency of lactase

- lactase

3 lactic acid + 3 CH4 + H2

(2)

Osmotic induction diarrhea

Galactosemia: an inherit disease

– galactose 1-phosphate uridyl transferase deficiency, diagnostic criterion for red blood cells

– diarrhea, liver enlargement, jaundice and cirrhosis, cataracts, lethargy, retarded mental development

– a delayed acquisition of language skills, ovarian failure for female patients

p. 452 There is a high incidence of cataract formation with age in populations that consume substantial amounts of milk into adulthood.

§ 16.2 The glycolytic pathways is tightly controlled

essentially irreversible reactions, three reactions

The methods of enzyme activity regulation allosteric effector ~ ms covalent phosphorylation ~ s transcription ~ h

A dual role of glycolysis:

generate ATP and provide building blocks, such as fatty acid synthesis

Skeletal muscle and liver regulation (Ch. 21)

–

F6PF1,6bisP

homotetramer

Glycolysis in muscle:

is controlled by energy charge

Phosphofructokinase is the most important control site in glycolysis

Phosphofructokinase – allosteric regulation

energy charge, ATP / AMP (, PFKase act. )

pH value ( pH focus at lactic acid PFKase act. )

(sigmoid)

(Hyperbolic)¤ [AMP] is positive

regulator

¤ adenylate kinase

2 ADP ATP + AMP

ATP is salvaged from ADP

¤ total adenylate pool is

constant

[ATP] [ADP] [AMP]

ex. 15Km

Glycolysis in muscle:

Hexokinase: is inhibited by its product, G6P

G6P fates (Ch. 20)

increase [G6P] imply:

no longer requires Glc for energy or for the synthesis of glycogen

Glc will be left in the blood

if phosphofructokinase is inhibited [F6P]

[G6P] hexokinase is inhibited

Pyruvate kinase:

is allosterically inhibited by ATP and alanine, former is related to energy

charge and latter is building blocks

Glycolysis in muscle:

Glycolysis in liver:

liver function: maintains blood-glucose level, the regulation is more

complex than muscle

Phosphofructokinase:

inhibited by citrate [TCA cycle] and enhancing the inhibitory effect of ATP

(not by pH of lactate)

activated by fructose 2,6-bisphosphate (F 2,6-BP)

[Glc] [F 2,6-BP] glycolysis [feedforward stimulation]

Phosphofructokinase – activated by fructose 2,6-bisphosphate

Glycolysis in liver:

liver function: maintains blood-glucose level

Glucokinase replace hexokinase

Glucokinase

is not inhibited by glucose 6-phosphate

provide glucose 6-phosphate for the synthesis of glycogen and

for the formation of fatty acid

its affinity for glucose is about 50-fold lower than that of hexokinase

brain and muscle first call on glucose when its supply is limited.

P. 456

– a tetramer of 57 kd subunits– isozymic forms: Liver (L) are controlled by reversible phosphorylation Muscle and brain (M)

Glucagon

cAMP

Protein kinase A

Allosteric inhibition

Glycolysis in liver:

Pyruvate kinase:

Isozymes contribute to the metabolic diversity of different organs

Normal serum-glucose level: 4~8 mMp. 457

Glucose transporters:

enable glucose to enter or leave animal cells

endurance exercise, GLUT4 No.

70-115 mg/100 ml

Hypoxia-inducible transcription factor (HIF-1)

– increase the expression of most glycolytic enzymes and glucose transporters

– increase the expression of vascular endothelial growth factor (VEGF)

angiogenic factors

Anaerobic exercise, activate HIF-1, ATP generation

Cancer stem cells

anoxia

Hypoxia vs. menstrual cycle HIF

Gluconeogenesis

is not a reversal of glycolysis

noncarbohydrate precursors of Glc, carbon skeleton

take place in liver, minor in kidney, brain, skeletal and heart muscle, to maintain the Glc level in the blood

Glc is the primary fuel of brain, and the only fuel of red blood cells

active skeletal muscle

protein breakdown

Triacylglycerol

hydrolysis

- 7.5 kcal/mol

0.7

-0.5

G°´

Glycolysis vs. Gluconeogenesis

¤ Three irreversible reactions, irrespective

Glycolysis:

hexokinase, phosphofructokinase, pyruvate kinase

Gluconeogenesis:

glucose 6-phosphatase, fructose 1,6-bisphosphatase,

pyruvate carboxylase, phosphoenolpyruvate carboxykinase

The stoichiometry of Glycolysis vs. Gluconeogenesis

¤ Glycolysis:

Glucose + 2 ADP + 2 Pi + 2 NAD+

2 Pyr + 2 ATP + 2 NADH + 2H+ + 2 H2O

G0’= - 20 kcal / mol

if reverse?

¤ Gluconeogenesis:

2 Pyr + 4 ATP + 2 GTP + 2 NADH + 6 H2O

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

G0’= - 9 kcal / mol

NTP hydrolysis is used to power an energetically unfavorable reaction

Both reactions are exergonic

Compartmental cooperation- mitochondrial

NADH-malate dehydrogenase

NAD+-malate dehydrogenase

Specific transporter

PEP + CO2

PEP carboxykinase

GTP

Pyruvate carboxylaseMito

G0’

decarboxylation

Pyruvate carboxylase (Pyr + CO2 + ATP + H2O OAA + ADP + Pi + 2 H+)

The only mitochondrial enzymes among the enzymes of gluconeogenesis

S

(PCase)

HCO3- + ATP HOCO2-PO3

2- + ADP carboxyphosphate: activated form of CO2

Biotin-Enz + HOCO2-PO3

2- CO2-biotin-Enz + Pi is activated by acetyl CoA (p. 493)

CO2-biotin-Enz + Pyr biotin-Enz + OAA

(ATP-activating domain, p. 711)

-amino group of Lys

Carbonic anhydrase

Free glucose generation

F1,6bisP F6P G6P ••• Glc

The endpoint of gluconeogenesis in most tissues,

can keep Glc or G6P is converted into glycogen.

In liver and to a lesser extent the kidney,

five proteins are involved

SP: a calcium-binding stabilizing protein

(Does not take place in cytoplasm)

Gluconeogenesis

Reciprocal control:Glycolysis and gluconeogenesis are not highly active at the same

time– Energy state– Intermedia: allosteric effectors – Regulators: hormones Amounts and activities of distinctive enzymes

Fed state:

insulin

low energy state

Starvation:

glucagon

rich in precursors

high energy state

p. 465

Biofunctional of phosphofructokinase 2phosphofructokinase / fructose bisphosphatase 2

F6P F2,6BisP

L (liver) / M (muscle) isoforms

a single 55-kd polypeptide chain

Janus

Fructose 2,6-bisphosphate: synthesis and degradation

In liver:

PEP carbokinase

F 1,6-bisphosphatase Glycolytic enzymes (pyruvate kinase)

The first irreversible reaction of glycolysis:Glc G6P

¤ Hexokinase: is inhibited by G6P

Km of sugars: 0.01 ~ 0.1 mM

Glucokinase: not inhibited by G6P

Km of glucose: ~10 mM

present in liver, to monitor blood-glucose level.

¤ Committed step

the most important control step in the pathway

G6P glycogen biosynthesis

fatty acid biosynthesis

pentose phosphate pathway

Hormones

¤ Affect the expression of the gene of the essential enzymes – change the rate of transcription – regulate the degradation of mRNA¤ allosteric control (~ms); phosphorylation control (~ s); transcription control (~ h to d)

The promoter of the PEP carboxykinase (OAAPEP) gene

IRE: insulin response element;

GRE: glucocorticoid response element

TRE: thyroid response element

CRE: cAMP response element

Substrate cycle (futile cycle)

Biological significancesSimultaneously fully active(1) Amplify metabolic

signals(2) Generate heat bumblebees: PFKase F1,6-bisPTase: is not inhibited by AMP

honeybees:only PFKase (02)

If 10

malignant hyperthermia

Cori cycle:

carriers

+ NADH

+ NAD+

AlaAla

Ala metabolism:

maintain nitrogen balance

transaminase

Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize and release glucose

Pyr LactateAbsence of O2

Well-oxygenatedTCA cycle

Integration of glycolysis and gluconeogenesis during a sprint

Lactate dehydrogenase

¤ a tetramer of two kinds of 35-kd subunits encoded by similar genes

¤ H type: in heart (muscle)

M type: in skeletal muscle and liver

¤ H4 isozyme (type 1): high affinity for lactate, lactatepyruvate,

under aerobic condition

H3M1 isozyme (type 2)

H2M2 isozyme (type 3)

H1M3 isozyme (type 4)

M4 isozyme (type 5): pyruvate lactate under anaerobic condition a series of homologous enzymes, foster metabolic cooperation between organs.

Biotin: abundant in some foods and is synthesized by intestinal bacteria

Avidin (Mr 70,000): rich in raw egg whites/a defense function

              

        

                                             

The Biotin-Avidin System can improve sensitivity because of

the potential for amplification due to multiple site binding.

Purification

Ex. 11

96T2

96T3

97T

97T

98T

98T

98T

96C

97C