Biochemical Energetics

8/9/2019 Biochemical Energetics

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Biochemical Energetics

Copyright 1999-2004 by Joyce J. Diwan.

All rights reserved.

Biochemistry of Metabolism


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Free energy of a reaction

The free energy change ((G) of a reaction determines

its spontaneity. A reaction is spontaneous if(G is

negative (if the free energy of products is less than that

of reactants).

(Go' = standard free energy change (at pH 7, 1M

reactants & products); R= gas constant;T = temp.

For a reaction A + B C + D

(G = (Go

' + RT ln[C][D]

[A][B]


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(Go'of a reaction may be positive, & (G negative,

depending on cellular concentrations of reactants and

products.

Many reactions for which (Go' is positive arespontaneous because other reactions cause depletion of

products or maintenance of high substrate concentration.

For a reaction

G G' RT l[A][ ]

[ ][ ]


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At equilibrium

(G = 0.

K'eq, the ratio

[C][D]/[A][B] atequilibrium, is the

equilibrium constant.

An equilibrium constant(K'eq) greater than one

indicates a spontaneous

reaction (negative (Gr').

(G (G' RT l

(G' RT l

(G' -RTl

de ining K'e

(G' -RT l K 'e

[C][ ][A][ ]

[C][ ]

[A][ ]

[C][ ][A][ ]

[C][ ]

[A][ ]


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K'e(G

'

kJ/mol

Starting ith 1 reactants

products, the reaction:

104

- 23 proceeds or ar d (spontaneous)

102

-11 proceeds or ar d (spontaneous)

100

1 0 is at e ili ri m

10-2 11 reverses to orm reactants

10-4 + 23 reverses to orm reactants

(Go' = RT ln K'eq

Variation of equilibrium constant with (Go (25 oC)


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Energy coupling

A spontaneous reaction may drive a non-spontaneous

reaction.

Free energy changes of coupled reactions are additive.

A. ome enzyme-catalyzed reactions are interpretable as

two coupled half-reactions, one spontaneous and the

other non-spontaneous.

At the enzyme active site, the coupled reaction is

kinetically facilitated, while individual half-reactions

are prevented.

Free energy changes of half reactions may be summed,

to yield the free energy of the coupled reaction.


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For example, in the reaction catalyzed by the Glycolysis

enzyme Hexokinase, the half-reactions are:

ATP + H2O m ADP + Pi (Go' =31 kJ/mol

Pi + glucosem glucose-6-P + H2O (Go' = +14 kJ/mol

Coupled reaction:

ATP + glucose mADP + glucose-6-P (Go' =17 kJ/mol

The structure of the enzyme active site, from which H2Ois excluded, prevents the individual hydrolytic reactions,

while favoring the coupled reaction.


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B. Two separate reactions, occurring in the same cellularcompartment, one spontaneous and the other not, may be

coupled by a common intermediate (reactant or product).A hypothetical, but typical, example involving PPi:

Enzyme 1:

A + ATP m B + AMP + PPi (Go' = + 15 kJ/mol

Enzyme 2:PPi + H2O m 2 Pi (G

o' = 33 kJ/mol

Overall spontaneous reaction:

A + ATP + H2O m B + AMP + 2 Pi (Go' = 18 kJ/mol

Pyrophosphate (PPi) is often the product of a reactionthat needs a driving force.

Its spontaneous hydrolysis, catalyzed by Pyrophosphataseenzyme, drives the reaction for which PPi is a product.


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Energy coupling in ion transport

Ion Transport may be

coupled to a chemical

reaction, e.g., hydrolysis orsynthesis of ATP.

In this diagram & below,

water is not shown. It should

be recalled that the ATP

hydrolysis/synthesis reaction

is: ATP + H2Om ADP + Pi.

S1 S2

ATP

ADP + Pi

Si e 1 Si e 2


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The free energy change (electrochemical potential

difference) associated with transport of an ionS

acrossa membrane from side 1 to side 2 is:

R= gas constant, T = temperature, Z = charge on the ion,

F =Faraday constant, (= = voltage.

(G R T l + Z F (=

[S]1

[S]2

S1 S2

Side 1 Side 2


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(G for ion flux - varies with ion gradient & voltage. (G for chemical reaction - negative(Go' for ATP

hydrolysis;(G depends also on [ATP], [ADP], [Pi].

ince free energy changes

are additive, the

spontaneous direction

for the coupled reactionwill depend on relative

magnitudes of:

S1 S2

ATP

ADP + Pi

Side 1 Side 2


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Two examples:

Active Transport: pontaneous ATP hydrolysis

(negative(

G) is coupled to (drives) ion flux against a

gradient (positive (G).

ATPsynthesis: pontaneous H+ flux (negative (G) is

coupled to (drives) ATP synthesis (positive (G).

S1 S2

ATP

ADP + Pi

active

tra sport

H+

1 H+

2

ATP

ADP + Pi

ATP

sy thesis


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N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPOP-O

O

O- O-

O O

O-

adenine

ribose

ATP

adenosine triphosphate

phosphoanhydride

bonds (~)

Phosphoanhydride linkages are said to be "high energy"

bonds. Bond energy is not high, just (G of hydrolysis.

"High energy" bonds are represented by the "~" symbol.

~P represents a phosphate group with a large negative (G

of hydrolysis.


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Compounds with high energy bonds are said to

have high group transfer potential.

For example, Pi may be spontaneously cleaved from

ATP for transfer to another compound, e.g., to a

hydroxyl on glycerol.

High energy bonds


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Potentially, 2 ~P bonds can be cleaved, as 2 phosphates

are released by hydrolysis from ATP.

AMP~P~P AMP~P + Pi (ATP ADP + Pi)

AMP~P AMP + Pi (ADP AMP + Pi)

Alternatively:

AMP~P~P AMP + P~P (ATP AMP + PPi)

P~P 2 Pi (PPi 2Pi)


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Example: AMPPNP.

uch analogs have been used to study the dependence of

coupled reactions on ATP hydrolysis.

In addition, they have made it possible to crystallize anenzyme that catalyzes ATP hydrolysis with an ATPanalog at the active site.

AMPPNP (ADPNP) ATP analog

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPNP-O

O

O- O-

O O

O-

H

Artificial ATPanalogs have

been designedthat are resistantto cleavage ofthe terminal

phosphate byhydrolysis.


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Inorganic polyphosphate

Many organisms store energy as inorganicpolyphosphate, a chain of many phosphate residueslinked by phosphoanhydride bonds:

P~P~P~P~P...

Hydrolysis of Pi residues from polyphosphate may becoupled to energy-dependent reactions.

Depending on the organism or cell type, inorganicpolyphosphate may have additional functions.

E.g., it may serve as a reservoir for Pi, a chelator ofmetal ions, a buffer, or a regulator.


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Why do phosphoanhydride linkages have a high (G

of hydrolysis? Contributing factors for ATP & PPiinclude:

Resonance stabilization of products of hydrolysis

exceeds resonance stabilization of the compound

itself.

Electrostatic repulsionbetween negativelycharged phosphate oxygen atoms favors

separation of the phosphates.


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Creatine Kinase catalyzes the reversible reaction:

Phosphocreatine + ADPm ATP + creatine

Phosphocreatine is produced when ATP levels are high.

During exercise in muscle, phosphate is transferred fromphosphocreatine to ADP, to replenish ATP.

Phosphocreatine may also be used to transport ~P fromone compartment of a cell to another.

O P

H

N C

O

O

N

NH2+

CH2

CH3

C

O

O

phosphocreatine

Phosphocreatine (creatinephosphate), anothercompound with a "highenergy" phosphate linkage,is used in nerve & muscleforstorage of ~P bonds.


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A reaction important for equilibrating ~P amongadenine nucleotides within a cell is that catalyzed by

Adenylate Kinase:ATP + AMPm 2 ADP

The Adenylate Kinase reaction is also important becausethe substrate for ATP synthesis, e.g., by mitochondrial

ATP ynthase, is ADP, while some cellular reactionsdephosphorylate ATP all the way to AMP.

The enzyme Nucleoside Diphosphate Kinase (NuDiKi)equilibrates ~P among the various nucleotides that areneeded, e.g., for synthesis of DNA & RNA.

NuDiKi catalyzes reversible reactions such as:

ATP + GDPm ADP + GTP,

ATP + UDPm ADP + UTP, etc.


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Phosphoenolpyruvate (PEP), involved in ATP synthesisin Glycolysis, has a very high (G of Pi hydrolysis.

Removal of Pi from ester linkage in PEP is spontaneousbecause the enol spontaneously converts to a ketone.

The ester linkage in PEP is an exception.

C

C

O O

OPO32

CH2

C

C

O O

O

CH3

C

C

O O

OH

CH2

ADP ATP

H+

PEP enolpyruvate pyruvate


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Generally phosphate esters (formed by splitting out

water between a phosphoric acid and an OH group) havea low but negative (Gof hydrolysis. Examples:

the linkage between the first phosphate of ATP & theribose hydroxyl

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPOP-O

O

O- O-

O O

O-

adenine

ribose

ATP(adenosine triphosphate)

ester linkage


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Other examples ofphosphate esters with low but

negative (G of hydrolysis: the linkage between phosphate & a hydroxyl group

in glucose-6-phosphate orglycerol-3-phosphate.

glycerol-3-phosphate

CH2

CH

CH2

OH

HO

O P

O

O

O

H O

OH

H

OHH

OH

CH2

H

OH

H

1

6

5

4

3 2

O P

O

OH

OH

glucose-6-phosphate


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ATP has special roles in energy coupling & Pi transfer.

(G of phosphate hydrolysis from ATP is intermediate

among examples below.ATP can thus act as a Pi donor, & ATP can be synthesizedby Pi transfer, e.g., from PEP.

Compound(G

o'of phosphate

hydrolysis, kJ/mol

Phosphoenolpyruvate (PEP)

Phosphocreatine

Pyrophosphate

ATP (to ADP)

Glucose-6-phosphate

Glycerol-3-phosphate


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Coenzyme A-SH + HO C

O

R

Coenzyme A-S C

O

R + H2O

A thioester forms between a carboxylic acid & a thiol

( H), e.g., the thiol ofcoenzyme A (abbreviated CoA- H).

Thioesters are ~ linkages. In contrast to phosphate esters,

thioesters have a large negative (G of hydrolysis.

ome otherhigh energy

bonds:


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The thiol of coenzyme A can react with a carboxyl groupof acetic acid (yielding acetyl-CoA) or a fatty acid

(yielding fatty acyl-CoA).The spontaneity of thioester cleavage is essential to therole of coenzyme A as an acyl group carrier.

Like ATP, CoA has a high group transfer potential.

Coenz e - H + HO C

O

CH3

Coenz e - C

O

CH3 + H2O

acetic acid

acetyl-CoA


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Coenzyme A includes

F-mercaptoethylamine,in amide linkage to the

carboxyl group of the B

vitamin pantothenate.

The hydroxyl ofpantothenate is in ester

linkage to a phosphate

ofADP-3'-phosphate.

The functional group is

the thiol ( H) of

F-mercaptoethylamine.

N

NN

N

NH2

O

OHO

HH

H

CH2

H

OPOPOH2C

O

O O

O

P

O

O

O

C

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

F-mercaptoethylamine

pantothenate

P-3 -phosphate

oenzyme


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3',5'-Cyclic AMP (cAMP), is usedby cells as a transient signal.

Adenylate Cyclase catalyzes cAMPsynthesis: ATPp cAMP + PPi.

The reaction is highly spontaneous

due to the production ofPPi, whichspontaneously hydrolyzes.

Phosphodiesterase catalyzeshydrolytic cleavage of one Pi ester(red), converting cAMPp 5'-AMP.

N

NN

N

NH2

O

OHO

HH

H

H2C

HO

PO

O-

1'

3'

5' 4'

2'

cAMP

This is a highly spontaneous reaction, because cAMP issterically constrained by having a phosphate with esterlinks to 2 hydroxyls of the same ribose. The lability ofcAMP to hydrolysis makes it an excellent transient signal.


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List compounds exemplifying the following rolesof "high energy" bonds:

Energy transfer or storage

ATP, PPi, polyphosphate, phosphocreatine

Group transfer

ATP, Coenzyme A

Transient signal

cyclic AMP


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Oxidation & reduction

Oxidation of an iron atom involves loss of an electron

(to an acceptor): Fe++ (reduced) Fe+++ (oxidized) + e-

Since electrons in a C-O bond are associated more with

O, increased oxidation of a C atom means increased

number of C-O bonds. Oxidation of C is spontaneous.

Increasing oxidation of carbon

H

CH H

H

H

CH OH

H

H

C

H

O

O

C

O

OH

C

H

O


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NAD+, NicotinamideAdenine Dinucleotide,

is an electronacceptorin catabolic pathways.

The nicotinamide ring,derived from the

vitamin niacin, accepts2 e- & 1 H+ (a hydride)in going to the reducedstate, NADH.

NADP+/NADPH issimilar except for Pi.NADPH is e donor insynthetic pathways.

H

C NH2

O

CH2

H

N

HOH OH

H H

OOP

O

HH

OH OH

H H

OCH2

N

N

N

NH2

OP

O

O

O

+

NO

nicotinamide

adenine

esterified toPi in NADP

+

Nicotinamide

AdenineDinucleotide


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NAD+/NADH

The electron transfer reaction may be summarized as :NAD+ + 2e + H+m NADH.

It may also be written as:

NAD+ + 2e + 2H+m NADH + H+

N

R

H

C

NH2

O

N

R

C

NH2

OH H

+

2

e

+

H+

NAD+

NADH


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FAD (Flavin Adenine Dinucleotide), derived from the

vitamin riboflavin, functions as an e acceptor. Thedimethylisoalloxazine ring undergoes reduction/oxidation.

FAD accepts 2 e- + 2 H+ in going to its reduced state:

FAD + 2 e- + 2 H+m FADH2

C

CC

H

C

C

H

C

NC

C

N

NC

NH

C

H3C

H3C

O

O

CH2

HC

HC

HC

H2C

OH

O P O P O

O

O-

O

O-

Ribose

OH

OH

Adenine

C

CC

H

C

C

H

C

NC

C

H

N

N

H

C

NH

C

H3C

H3C

O

O

CH2

HC

HC

HC

H2C

OH

O P O P O

O

O-

O

O-

Ribose

OH

OH

AdenineFAD FADH2

2 e + 2 H+

dimethylisoalloxazine


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NAD+ is a coenzyme, that reversibly binds to

enzymes.

FAD is a prosthetic group, that remains tightly

bound at the active site of an enzyme.

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Biochemical Energetics