Basic principles of metabolic fluxes

Fernando Rodrigues & Ricardo Silvestre

2022
Basic principles of metabolic fluxes

Outline

Energy Systems
Metabolism Basics
Glycolysis
Gluconeogenesis
Citric Acid Cycle
Lipid metabolism
Pentose Phosphate Pathway
Glycogen Metabolism
Amino Acid Metabolism
Introduction to alosteric metabolic regulation
Overview of Metabolism
The sum of all reactions that take place in a living organism

Metabolism = Catabolism + Anabolism

• Catabolism - larger molecules are broken down into smaller ones in a

process that usually releases energy
• Anabolism - larger molecules are made from small ones in a process the
usually requires energy
 Cell as a metabolic black box
Inputs Outputs

Outputs Inputs

DECONSTRUCTING CELULAR METABOLISM

Energy transfer: oxidation reduction

High energy

Low energy

Cells conserve energy in the form of ATP by coupling its synthesis to the release of energy via oxidation-
reduction (redox) reactions, where electrons are passed from an electron donor to an electron acceptor.

Activated carriers of electrons

Nicotinamide Adenine Dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+)

Flavin Adenine Dinucleotide (FAD)

Electron Transfers Release Large Amounts of Energy
Anabolism VS Catabolism

NADH has a strong tendency to donate electrons;

because its electrons are held in a high-energy linkage, the free-energy change for passing its
electrons to many other molecules is favorable
Flavin Adenine Dinucleotide Activated carriers of electrons

dimethylisoalloxazine O O
H H H
H3C C
C
C
N
C
C
NH 2 e + 2 H+ H3C C
C
C
N
C
C
NH

H3C C C C C O H3C C C C C O
C N N C N N
H H H
CH2 CH2

HC OH HC OH

HC OH HC OH
FAD Adenine
FADH2
HC OH O O HC OH O O Adenine

H2C O P O P O Ribose H2C O P O P O Ribose

O- O- O- O-

 NAD+ and NADP+ are coenzymes, that reversibly binds to enzymes

(soluble).
 FAD is a prosthetic group, that remains tightly bound at the active site of
an enzyme.
Chemical reactions What can we say about the reaction rate?

Modeladores de atividade
Chemical reactions Coupled reactions

C
B

B A D

A
The power bank of the cell
High energy is needed to
maintain the phosphate
bonds

Hydrolysis of ATP

The molecule acts to couple the energy of exergonic and endergonic processes,
making energetically unfavorable chemical reactions able to proceed.
 ATP hydrolysis
ATP production and consumption

Hexokinase, the half-reactions are:

ATP + H2O  ADP + Pi DGo' = 31 kJ/mol
Pi + glucose  glucose-6-P + H2O DGo' = +14 kJ/mol
Coupled reaction:
ATP + glucose  ADP + glucose-6-P DGo' = 17 kJ/mol
ATP production and consumption

ATP synthesis
Substrate-level phosphorylation

a substrate molecule donates its high energy P to ADP

making ATP

Oxidative phosphorylation

is the synthesis of ATP through an electrochemical

transmembrane gradient coupled to as an electron
transfer chain driven by substrate oxidation
 ATP synthesis

Mitochondria
The powerhouse

Doi: 10.1091/mbc.E15-03-0181
Key concepts Electron associated energy work
Oxidation-Reduction Reactions

work
heat Electron donor
work

Free energy
heat
work

Oxidation-Reduction
Reactions
 Oxidative phosphorilation

https://www.cusabio.com/pathway/Oxidative-Phosphorylation.html
Mobile electrons carriers Quinone cycle

ROS production
source of electrons
source of electrons
electron-transport chain
work

Free energy
heat Electron donor
work
heat
work

Oxidation-
Reduction
Reactions

Proton transport
The electrochemical proton gradient

energy
 Mitochondria

https://doi.org/10.3390/antiox10040520
Compartmentalization of carbon metabolism
Transport mechanisms

Key regulation of metabolic processes

Pathways, Energy, and Coupled Reactions
Metabolic reactions are arranged in pathways
The product of one reaction is the substrate for the next reaction in the pathway.
There are different topologies for metabolic pathways.
Common types of biochemical Pathways
How do we study/see/learn metabolic pathways?

Cell
How metabolism can be regulated?
- Substrate concentration

H H
HD B Ci i B iHi - Enzyme concentration

HJ G AH DhG EYAHC h Y
X
E J
n I V H H
E H E Kinetic parameters

HS i H S I h Ti h T
h X Vmax
Km Activators

h HU
nÇ VHÇ Inhibitors

Cell Pathways connectors

Molecules Connecting the different Pathways

-Substrates
-Products
-ATP+ADP+AMP
-NADH+NAD+
-FADH2+FAD

NADH NAD+ NADH

NADH

NAD+ NAD+
Metabolic fluxes
Equilibrium
Schematic Representation
Le Châtelier's principle

NADH NAD+
NAD+
NAD+
NADH
NADH
NADH

iii hh
h

VV
V SSS TTTT YYY ÇÇÇ
UUU
X
X
X So NADH Ç X

NADH Ç X
Metabolic fluxes
i
E G
H
J
I
n
NADH NAD+
h i

V S T Y Ç

What happen when V increases?

Types of metabolic regulation
Enzyme mediated
Metabolic pathways

• Catabolism - larger molecules are

broken down into smaller ones in a
process that usually releases energy
• Anabolism - larger molecules are
made from small ones in a process the
usually requires energy
Metabolic pathways
Catabolism VS Anabolism
Metabolic pathways

Metabolic remodeling
Central carbon metabolism
http://www.elmhurst.edu/~chm/vchembook/images/590metabolism.gif.
 Glucose metabolism
1st step Glucose transport
 Glycolysis
• Glycolysis is a series of 10 coupled reactions
• The pathway starts with glucose that comes into a cell hexokinase
from the extracellular compartment and is
immediately phosphorylated to glucose-6-phosphate.
Why? phosphoglucose
isomerase
•The phosphorylation traps the glucose in the cell.

phosphofructokinase

• The pathway then goes on to

•No return! aldolase

phosphorylate glucose-6-
phosphate into glucose-1,6- triosephosphate
isomerase

bisphosphate.
 glyceraldehyde
The pathway then goes on to split (lyse) 3-phosphate
the the 6-carbon glucose molecule into dehydrogenase

two 3-carbon molecules and to oxidize

these to α-keto acids (Pyruvate.
phosphoglycerate
kinase

The energy released in the pathway is used to phosphoglycerate

produce two types of energy rich molecules: mutase

ATP
enolase

NADH/H+
pyruvate
kinase
Glycolytic intermediates funnel into biosynthetic pathways
Regeneration of NAD+ by LDH or mitochondria

Metabolic flux
Glycolytic flux

Ratio
Lactate/glucose
Understanding the Warburg Effect: The Metabolic
Requirements of Cell Proliferation

Fermentation Vs anaerobic glycolysis

“cells preferentially use glycolysis

rather than oxidative phosphorylation
for energy production”

10.1126/science.1160809
Glycolysis and gluconeogenesis share many enzymes
From Cytosol to Mitochondria
Citric Acid Cycle
Role of the TCA cycle in anabolism
Overview of lipid metabolism
FATTY ACIDS CATABOLISM
 Overview of FA metabolism

Carbohydrate metabolism

ATP
PPP

?
NADPH

NADH
The pentose phosphate pathway

CO2
6

6 CO2
12NADPH
2
The pentose phosphate pathway
Glycogen metabolism

The Forgotten glucose

source

immunometabolism
Amino acid degradation
The central role of Glutamate
Introduction to alosteric metabolic regulation
Reciprocal regulation of glycolysis and gluconeogenesis
60

50 low [ATP]

PFK Activity
40

30

20
high [ATP]

10

0
0 0.5 1 1.5 2
[Fructose-6-phosphate] mM

doi: 10.1101/cshperspect.a040568
Fatty acid metabolism regulation