Biochemistry: Thermodynamics in metabolism

Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**

Consortium leader

PETER PAZMANY CATHOLIC UNIVERSITY

Consortium members

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***

**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben

***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg.

PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS

UNIVERSITY

Semmelweis University

ORGANIC AND BIOCHEMISTRY

Thermodynamics and kinetics of metabolic pathways

http://semmelweis-egyetem.hu/

(Szerves és biokémia)

(Metabolikus utak termodinamikája és kinetikája)

Krasimir Kolev

Biochemistry: Thermodynamics in metabolism

Lecture objectives

At the end of the presentation the participant will be able:

1) To define the subject of Biochemistry

2) To discuss the reductionist approach to understanding metabolism

3) To describe the connection between thermodynamics and directionality of metabolic pathways

4) To interpret the spontaneity of reactions in living organisms in terms of coupling-in-series and coupling-in-parallel of reactions

5) To define the terms near-equilibrium and non-equilibrium reactions 6) To understand the relation between thermodynamics and kinetics of metabolic pathways

7) To describe the structure of proteins

8) To list the basic steps in the catalytic mechanism of serine proteases 9) To discuss the structure/function relations in enzyme action

10) To interpret the action of protease inhibitors

Biochemistry: Thermodynamics in metabolism

The subject of Biochemistry^DEF: chemical transformations

occurring in living organisms

Specific features:

• moderate temperature

• standard pressure

• compartments

• open systems

Biochemistry: Thermodynamics in metabolism

Biochemistry at a glance Metabolic charts

http://www.iubmb-nicholson.org/

the road maps of chemical transformations in the cell

Thermodynamics and kinetics

the rules for substance fluxes (which one?

when? where? how?)

Biochemistry: Thermodynamics in metabolism

The reductionist approach^DEF to establish the rules

f r

v

A ←⎯⎯ ⎯⎯→ v B

Biochemistry: Thermodynamics in metabolism

Thermodynamic rules

the energy transformations determine the direction and spontaneity of the processes

Valid for:

• a single reaction

• a series of reactions (= metabolic pathway)

• transport processes

Biochemistry: Thermodynamics in metabolism

The first law of thermodynamics

(the law of energy conservation)

H q w Δ = −

enthalpy^DEF change

heat

work

Reaction ΔH⁰ (kJ/mol)

palmitic acid+ 23O₂ Æ 16CO₂+ 16H₂O -10024

glucose + 6O₂ Æ 6CO₂+ 6H₂O -2813

alanine + 3O₂ Æ 2.5CO₂+ 2.5H₂O + 0.5urea -1304

ATP + H₂O Æ ADP + P_i -21

Biochemistry: Thermodynamics in metabolism

Energy requirements of human subjects during different activities

Intensity Activity Mean energy

requirement (kJ/min)

basal activity rest 4.5

low office work,

driving

6 – 10

moderate housekeeping,

swimming

15 – 25

high digging, marathon

running

30 - 85

Biochemistry: Thermodynamics in metabolism

Heat production in the living organisms

glukóz + 6O

6H

O + 6CO

38ADP + 38Pi 38ATP + 38H

O

ΔH=-2813 kJ ΔH=798 kJ

the difference is released as heat

Biochemistry: Thermodynamics in metabolism

The second law of thermodynamics

(for spontaneous processes ΔS>0)

S q Δ = T

reactants environment 0

S S

Δ + Δ >

entropy^DEF =heat absorbed in a reversible reaction/ temperature K heat

temperature

Biochemistry: Thermodynamics in metabolism

Gibbs’ free energy

For reaction conditions when the only effect on the environment is the emission or absorption of heat:

environment

S H

T Δ = −Δ

reactants 0 G H T S

Δ = Δ − Δ <

the second law of thermodynamics takes the form:

Gibbs’ free energy (not a true form of energy, contains an entropy term)

Biochemistry: Thermodynamics in metabolism

Relation between free energy and equilibrium 1.

f r

v

A ←⎯⎯ ⎯⎯→ v B

0 [ ]

.ln [ ] G G RT B

Δ = Δ + A

free energy change

free energy change for standard conditions (1 M [reactant], pH 7.0, 25°C)

gas constant

0 [ ]

.ln .ln

[ ]

G RT B RT K

Δ = − A = −

0 Δ = G

Biochemistry: Thermodynamics in metabolism

At equilibrium

0 [ ]

0 .ln

[ ] G RT B

= Δ + A

Relation between free energy and equilibrium 2.

Biochemistry: Thermodynamics in metabolism

Relation between free energy and equilibrium 3.

[ ] [ ]

B Γ = A

Mass action ratio

.ln .ln .ln

G RT K RT RT

K

Δ = − + Γ = Γ

Biochemistry: Thermodynamics in metabolism

Interpretation of free energy changes in metabolism

0

.ln

G G RT

Δ = Δ + Γ

Reaction ΔG⁰ kJ/mol ΔG kJ/mol Type

hexokinase -20.9 -27.9 non-equilibrium

G6P isomerase +2.1 -1.3 near-equilibrium

PFK -17.1 -26.5 non-equilibrium

aldolase +23.0 -6.1 near-equilibrium

GA3PDH+PGA kinase +7.9 -11.5 near-equilibrium

phosphoglyceromutase +4.6 -0.5 near-equilibrium

enolase -3.3 -2.4 near-equilibrium

pyruvate kinase -24.7 -15.8 non-equilibrium

Thermodynamic characteristics of the glycolytic reactions in rat heart

Biochemistry: Thermodynamics in metabolism

Relation between kinetics and thermodynamics 1.

f r

v

A ←⎯⎯ ⎯⎯→ v B

f f

v = k A v _r = k A _r

At equilibrium

f

f r f r

r

B k

v v k A k B K

A k

= ⇒ = ⇒ = =

Relation between kinetics and thermodynamics 2.

Biochemistry: Thermodynamics in metabolism

r r r

f f f

v k B k

v k A k K

= = Γ = Γ

p f p

r

v v v

A ⎯⎯→ v B

⎯⎯→ ←⎯⎯ ⎯⎯→

Biochemistry: Thermodynamics in metabolism

Relation between kinetics and thermodynamics 3.

For a metabolic pathway in steady state:

f p p 1

r

p f r

f f f

v v v

v v v v

v v K v K

− Γ Γ

= − ⇒ = = ⇒ = −

Kinetic interpretation of near- and non-equilibrium reactions:

• near-equilibrium:

• non-equilibrium:

f p

v ≈ v

f p

v > v

Biochemistry: Thermodynamics in metabolism

Coupling-in-series in metabolism

1 2

E

E E

S ←⎯⎯ ←⎯⎯ ←⎯⎯ ⎯⎯→ ⎯⎯→ ⎯⎯→ A B P

0

2 2

.ln [ ] [ ] G G RT B

Δ = Δ + A

Reaction ΔG⁰ kJ/mol ΔG kJ/mol Type

hexokinase -20.9 -27.9 non-equilibrium

G6P isomerase +2.1 -1.3 near-equilibrium

PFK -17.1 -26.5 non-equilibrium

Biochemistry: Thermodynamics in metabolism

Coupling-in-parallel in metabolism

1.

M L

B A

X Y

X Y

ATP ADP

NAD NADH₂

NADP NADPH₂

coenzyme A acetyl coenzyme A

Biochemistry: Thermodynamics in metabolism

Coupling-in-parallel in metabolism 2.

glucose-6-phosphate glucose

P _i H ₂ O

0

[glucose 6 phosphate].[

] .ln [glucose].[ ]

G G RT H O

P Δ = Δ + − −

Δ

Δ

ADP ATP

H ₂ O P _i

Δ

Biochemistry: Thermodynamics in metabolism

Coupling-in-parallel in metabolism 3.

glucose-6-phosphate glucose

ATP ADP

Δ G

=-16.7 kJ/mol

Biochemistry: Thermodynamics in metabolism

Kinetic and thermodynamic structure of metabolic pathways

10,01 100 10,1

0,01 90 0,1

S ←⎯⎯ ⎯⎯⎯ ⎯ ←⎯⎯ ←⎯⎯ → ⎯⎯→ ⎯⎯→ A B P

0

[ ]

.ln [ ] G G RT y

Δ = Δ + x

Biological significance:

• non-equilibrium reactions (directionality, regulation)

• near-equilibrium reactions (reversibility)

Biochemistry: Enzyme kinetics in metabolism

Relation between kinetics and thermodynamics

• thermodynamics: spontaneity and direction of the process

• kinetics: rate of the process

activation energy^DEF

Biochemistry: Enzyme kinetics in metabolism

Factors decreasing the activation energy during enzyme action

• proximity of substrates

• spatial orientation

• forced positional strain

• additional interactions with functional groups

Biochemistry: Enzyme kinetics in metabolism

Structure of enzymes 1: Amino acids

Biochemistry: Enzyme kinetics in metabolism

Common amino acids

Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 2: Peptide bonds

Primary structure

Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 3: Higher levels of

protein organization

α-helix β-sheet

Secondary structure ^DEF

Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 4: Higher levels of

protein organization

Tertiary structure^DEF

Enzymes in action

(changes in conformation

)

Biochemistry: Enzyme kinetics in metabolism

hexokinase

glucose

substrate

transient state

products

Biochemistry: Enzyme kinetics in metabolism

Hydrolysis of peptide bonds

(general mechanism)

Biochemistry: Enzyme kinetics in metabolism

Catalytic mechanism of serine proteases

proteases catalyze the hydrolysis of peptide bonds in proteins

Energy stages in the course of reaction

Biochemistry: Enzyme kinetics in metabolism

A closer look at the active site

of chymotrypsin

Biochemistry: Enzyme kinetics in metabolism

Role of the separate amino acids in the active site

- the hydroxyl oxygen of Ser195 loses its hydrogen to His57

- the nucleophilic oxygen attacks the carbonyl C of the peptide bond

Biochemistry: Enzyme kinetics in metabolism

First transient tetrahedral state of the enzyme-substrate complex

The tetrahedral structure of the

transient enzyme-substrate complex is stabilized by two amide hydrogens coordinating the anionic oxygen in the oxyanion hole.

Energy stages in the course of reaction

Biochemistry: Enzyme kinetics in metabolism

The tetrahedral structure decomposes to an acyl-enzyme intermediate and a peptide with new N-terminal portion is released.

Cleavage of the peptide bond

Energy stages in the course of the reaction

Biochemistry: Enzyme kinetics in metabolism

Second transient tetrahedral state of the enzyme-substrate complex

- a water molecule enters and attacks the ester bond of the enzyme-substrate complex - a second tetrahedral structure is formed as one of the waters hydrogens is passed to His57

Biochemistry: Enzyme kinetics in metabolism

Cleavage of the ester bond linking the enzyme and the substrate

The tetrahedral structure decomposes releasing a product peptide with new C-terminal portion.

Ser195 recovers its hydrogen from His57 and the initial state of the enzyme is restored.

Energy stages in the course of the reaction

Biochemistry: Enzyme kinetics in metabolism

Mechanism of action of protease inhibitors

Most natural inhibitors are proteins which act as pseudosubstrates blocking the catalytic mechanism at different stages

1. Reversible inhibitors (e.g. pancreatic trypsin inhibitor): bind, but the first tetrahedral structure is not formed

protease

inhibitor

pseudosubstrate loop

Biochemistry: Enzyme kinetics in metabolism

Mechanism of action of protease inhibitors 2.

2. Irreversible inhibitors (e.g. serpins^DEF): the first tetrahedral structure (IP_M) and the acylated enzyme-substrate complex (IP_acyl) are formed, but the ester bond cannot be cleaved, because the catalytic site is distorted

Messages to take home

1) Biochemistry studies the chemical reactions in living organisms

2) The reductionist approach dissects metabolism to separate reactions and builds up the whole system from these pieces of knowledge

3) Thermodynamics determines the directionality of metabolic pathways and its principles are valid for each separate reaction as well as for sequences of reactions 4) Spontaneity of reactions under the concentration, temperature and pressure conditions of living organisms is maintained by their coupling-in-series and coupling-in-parallel

5) Near-equilibrium reactions have free energy change approaching zero, whereas non-equilibrium reactions have large negative free energy change

6) Proteins are composed of amino acids bound through peptide bonds and polypeptide chains form higher-order spatial structures

7) Specific interactions between amino acid residues in the active site of enzymes and the substrates result in a decrease in the activation energy of the catalyzed reaction (e.g. serine protease)

Biochemistry: Enzyme kinetics in metabolism

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 1

Which statement is true regarding the direction of the biochemical processes?

A. Spontaneous reactions are accompanied by positive changes in the free enthalpy.

B. Spontaneous reactions are accompanied by positive changes in the free entropy.

C. Spontaneous reactions are accompanied by positive changes in the free energy.

D. Spontaneous reactions are accompanied by negative changes in the free enthalpy.

E. Spontaneous reactions are accompanied by negative

changes in the free energy.

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 2

What is the maximal efficiency at which the energy released

from glucose oxidation is converted into energy that can be used as ATP?

A. 90 %

B. 75 %

C. 50 %

D. 30 %

E. 10 %

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 3

What is the relation between equilibrium and the change in the free energy of a reaction?

A. At equilibrium the free energy is not changed.

B. At equilibrium the free energy change is equal to the standard free energy change.

C. At equilibrium the free energy change is always positive.

D. At equilibrium the free energy change is always negative.

E. The term free energy change can be applied only for

reactions in equilibrium.

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 4

Which statement is true regarding reactions coupled in parallel in metabolic pathways?

A. The presence of cofactor is obligatory.

B. The presence of coenzyme is obligatory.

C. The presence of prosthetic group is obligatory.

D. The reactions are accompanied always by negative standard free energy change.

E. The sign of the change in the free energy is the same for the

coupled reactions (a reaction with positive change in the free

energy cannot be coupled-in-parallel to a reaction with negative

change).

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 5

Which statements are true regarding non-equilibrium reactions in metabolic pathways?

1. They determine the direction of the pathway.

2. They are targets of regulation.

3. They are reversible.

4. They confer reliability of the pathway.

5. They can participate in multiple pathways.

A. 1,2 B. 3,4 C. 4,5 D. 3,5 E. 2,4

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 6

Which statement is true regarding the role of enzymes in metabolic pathways?

A. They determine the direction of the reaction.

B. They determine the rate of the reaction.

C. They determine the free energy change of the reaction.

D. They determine the enthalpy change of the reaction.

E. None of the above

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 7

At body temperature proteins are stable molecules, because

A. their synthesis is accompanied by large negative free energy change.

B. their degradation is accompanied by large negative free energy change.

C. their hydrolysis requires high activation energy.

D. their hydrolysis requires no or only minimal activation energy.

E. their hydrolysis occurs only at significantly higher

temperatures.

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 8

Select the functional group which performs the nucleophilic attack in the course of the catalytic action of serine proteases.

A. carboxyl.

B. hydroxyl.

C. imidazole.

D. sulfhydryl.

E. none of the above.

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 9

Select the type of bond transiently formed between the substrate and the enzyme in the course of the catalytic action of serine

proteases.

A. ether.

B. ester.

C. peptide.

D. amide.

E. acidic anhydrate.

Biochemistry: Enzyme kinetics in metabolism Comprehension problem 10

Which statement is true regarding the function of protease inhibitors?

A. Only the structure of the enzyme is changed during the inhibition of the protease.

B. Only the structure of the inhibitor is changed during the inhibition of the protease

C. The structure of both enzyme and inhibitor is changed during the inhibition of the protease.

D. Following the inhibition of the protease active inhibitor can always be released from the complex.

E. The inhibition of the protease is always reversible.

Recommended literature

Orvosi Biokémia (Ed. Ádám Veronika): pp. 21-30, 55-60

Biochemistry: Enzyme kinetics in metabolism

Biochemistry: Enzyme kinetics in metabolism

Answers to comprehension problems

1. E; 2. D; 3. A; 4. A; 5. A; 6. B; 7. C; 8. B; 9. B; 10. C