Biochemical Energetics

Biochemical Energetics

What is energy ? The capacity for doing work. Laws of Thermodynamics = laws of energy Zeroth law If two thermodynamic systems are each in thermal equilibrium with a third, then they are in thermal equilibrium with each other First law Energy can neither be created nor destroyed. It can only change forms. In any process in an isolated system, the total energy remains the same. Second law Energy systems have a tendency to increase their entropy rather than decrease it.

Enthalpy (H) - equals Energy plus Work at constant pressure which is the same as the change in heat at constant pressure ΔH – mean variation of entalpy in a system and can be used in biochemistry to see if a reaction it is thermodynamic posible. Entropy (S) – means the rate of disorder in a system ΔS – show how a system can be transformed in a more ordered or disordered one. Temperature T – is a measure of thermal agitation. It is expresed only in Kelvin (K). The minimum T can not be lower than 0 K (273,16 Celsius). ΔT – variation of temperature in a system. ΔG – means free energy. This variation of energy in a biochemical system predict the direction of reactions.

Free energy of a reaction The free energy change (DG) of a reaction determines its spontaneity. A reaction is spontaneous if DG is negative (if the free energy of products is less than that of reactants). For a reaction A + B  C + D [C] [D] DG = DG ' + RT ln [A] [B] o

DGo' = standard free energy change (at pH 7, 1M reactants & products); R = gas constant; T = temp.

For a reaction A + B  C + D [C] [D] DG = DGº' + RT ln [A] [B]

DGo' of a reaction may be positive, & DG negative, depending on cellular concentrations of reactants and products. Many reactions for which DGo' is positive are spontaneous because other reactions cause depletion of products or maintenance of high substrate concentration.

At equilibrium DG = 0. K'eq, the ratio [C][D]/[A][B] at equilibrium, is the equilibrium constant. An equilibrium constant (K'eq) greater than one indicates a spontaneous reaction (negative DG').

[C] [D] DG = DGº' + RT ln [A] [B] [C] [D]  = DGº' + RT ln [A] [B] [C] [D] DGº' = - RTln [A] [B] [C] [D] defining K'eq = [A] [B] DGº' = - RT ln K'eq

DGo' = - RT ln K'eq Variation of equilibrium constant with DGo‘ (25 oC)

K'eq

DG º' kJ/mol

Starting with 1 M reactants & products, the reaction:

10

4

- 23

proceeds forward (spontaneous)

10

2

- 11

proceeds forward (spontaneous)

100 = 1 10 10

0

is at equilibrium

-2

+ 11

reverses to form “reactants”

-4

+ 23

reverses to form “reactants”

Energy coupling  A spontaneous reaction may drive a non-spontaneous reaction.  Free energy changes of coupled reactions are additive. A. Some 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.

For example, in the reaction catalyzed by the Glycolysis enzyme 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 The structure of the enzyme active site, from which H2O is excluded, prevents the individual hydrolytic reactions, while favoring the coupled reaction.

B. Two separate reactions, occurring in the same cellular compartment, 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  B + AMP + PPi DGo' = + 15 kJ/mol Enzyme 2: PPi + H2O  2 Pi DGo' = – 33 kJ/mol Overall spontaneous reaction: A + ATP + H2O  B + AMP + 2 Pi DGo' = – 18 kJ/mol Pyrophosphate (PPi) is often the product of a reaction that needs a driving force. Its spontaneous hydrolysis, catalyzed by Pyrophosphatase enzyme, drives the reaction for which PPi is a product.

Energy coupling in ion transport Ion Transport may be coupled to a chemical reaction, e.g., hydrolysis or synthesis of ATP.

In this diagram & below, water is not shown. It should be recalled that the ATP hydrolysis/synthesis reaction is: ATP + H2O  ADP + Pi.

ADP + Pi

S2

S1 ATP Side 1

Side 2

S1

S2 Side 1

Side 2

The free energy change (electrochemical potential difference) associated with transport of an ion S across a membrane from side 1 to side 2 is:  [S]2 DG = R T ln + Z F DY [S]1

R = gas constant, T = temperature, Z = charge on the ion, F = Faraday constant, DY = voltage.

Since free energy changes are additive, the spontaneous direction for the coupled reaction will depend on relative magnitudes of:

ADP + Pi

S2

S1 ATP Side 1

Side 2

 DG for ion flux - varies with ion gradient & voltage.  DG for chemical reaction - negative DGo' for ATP hydrolysis; DG depends also on [ATP], [ADP], [Pi].

ADP + Pi

ADP + Pi

S2

S1 active transport

ATP

+

+

H

H

1

ATP synthesis

2

ATP

Two examples:

Active Transport: Spontaneous ATP hydrolysis (negative DG) is coupled to (drives) ion flux against a gradient (positive DG). ATP synthesis: Spontaneous H+ flux (negative DG) is coupled to (drives) ATP synthesis (positive DG).

“High energy” bonds NH 2

ATP adenosine triphosphate

O -O

P O-

O O

P O-

N

N

O O

P

O

phosphoanhydride bonds (~)

N

adenine

CH2

O-

N

O

H

H

OH

H OH

H

ribose

Phosphoanhydride bonds (formed by splitting out H2O between 2 phosphoric acids or between carboxylic & phosphoric acids) have a large negative DG of hydrolysis.

NH 2

ATP adenosine triphosphate

O -O

P O-

O O

P

adenine N

N

O O

O-

phosphoanhydride bonds (~)

P

N

O

CH 2

O-

O

H

H

OH

H OH

H

N

ribose

Phosphoanhydride linkages are said to be "high energy" bonds. Bond energy is not high, just DG of hydrolysis. "High energy" bonds are represented by the "~" symbol. ~P represents a phosphate group with a large negative DG of hydrolysis. Mangesium ions are key factors.

“High energy” bonds 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 group on glucose).

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)

 ATP often serves as an energy source. Hydrolytic cleavage of one or both of the "high energy" bonds of ATP is coupled to an energy-requiring (non-spontaneous) reaction. (Examples presented earlier.)  AMP functions as an energy sensor & regulator of metabolism.

When ATP production does not keep up with needs, a higher portion of a cell's adenine nucleotide pool is AMP. AMP stimulates metabolic pathways that produce ATP.

• Some examples of this role involve direct allosteric activation of pathway enzymes by AMP. • Some regulatory effects of AMP are mediated by the enzyme AMP-Activated Protein Kinase.

NH 2

Artificial ATP analogs have been designed that are resistant to cleavage of the terminal phosphate by hydrolysis.

N

-O

O

H

O

P

N

P

O-

O-

N

O O

P

O

CH 2

OH

N N

O

H

H

OH

H OH

AMPPNP (ADPNP) ATP analog

Example: AMPPNP. Such analogs have been used to study the dependence of coupled reactions on ATP hydrolysis. In addition, they have made it possible to crystallize an enzyme that catalyzes ATP hydrolysis with an ATP analog at the active site.

A reaction important for equilibrating ~P among adenine nucleotides within a cell is that catalyzed by Adenylate Kinase: ATP + AMP  2 ADP The Adenylate Kinase reaction is also important because the substrate for ATP synthesis, e.g., by mitochondrial ATP Synthase, is ADP, while some cellular reactions dephosphorylate ATP all the way to AMP.

The enzyme Nucleoside Diphosphate Kinase (NuDiKi) equilibrates ~P among the various nucleotides that are needed, e.g., for synthesis of DNA & RNA. NuDiKi catalyzes reversible reactions such as: ATP + GDP  ADP + GTP, ATP + UDP  ADP + UTP, etc.

Inorganic polyphosphate Many organisms store energy as inorganic polyphosphate, a chain of many phosphate residues linked by phosphoanhydride bonds: P~P~P~P~P... Hydrolysis of Pi residues from polyphosphate may be coupled to energy-dependent reactions.

Depending on the organism or cell type, inorganic polyphosphate may have additional functions. E.g., it may serve as a reservoir for Pi, a chelator of metal ions, a buffer, or a regulator.

Why do phosphoanhydride linkages have a high DG of hydrolysis? Contributing factors for ATP & PPi include:  Resonance stabilization of products of hydrolysis exceeds resonance stabilization of the compound itself.  Electrostatic repulsion between negatively charged phosphate oxygen atoms favors separation of the phosphates.

Phosphocreatine (creatine phosphate), another compound with a "high energy" phosphate linkage, is used in nerve & muscle for storage of ~P bonds.

O -

O

CH3 H N

P O

-

C

N

O CH2

NH2+

C O-

phosphocreatine

Creatine Kinase catalyzes: Phosphocreatine + ADP  ATP + creatine This is a reversible reaction, though the equilibrium constant slightly favors phosphocreatine formation.  Phosphocreatine is produced when ATP levels are high.  When ATP is depleted during exercise in muscle, phosphate is transferred from phosphocreatine to ADP, to replenish ATP.

O-

O C C

CH2

PEP

O-

O ADP ATP

OPO32H+

C

C C

O-

O

OH

CH2

enolpyruvate

C

O

CH3

pyruvate

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

Removal of Pi from ester linkage in PEP is spontaneous because the enol spontaneously converts to a ketone. The ester linkage in PEP is an exception.

NH2 N

N

ester linkage O -O

P O-

O O

P O-

N

O O

P

O

CH2

O-

ATP (adenosine triphosphate)

adenine O

H

H

OH

H OH

H

N

ribose

Generally phosphate esters, formed by splitting out water between a phosphoric acid and an OH group, have a low but negative DG of hydrolysis. Examples:  the linkage between the first phosphate and the ribose hydroxyl of ATP.

O 6 CH

2

4

OH

P OH O

5

H

O

H OH 3

H

OH

CH2 H

CH

O

1

H 2

HO

OH

OH

OH

glucose-6-phosphate

CH2

O

P

O-

O-

glycerol-3-phosphate

Other examples of phosphate esters with low but negative DG of hydrolysis:  the linkage between phosphate & a hydroxyl group in glucose-6-phosphate or glycerol-3-phosphate.

O

Protein Kinase OH + ATP

Protein

Protein

O

P

O- + ADP

OPi

H2O

Protein Phosphatase

 the linkage between phosphate and the hydroxyl group of an amino acid residue in a protein (serine, threonine or tyrosine). Regulation of proteins by phosphorylation and dephosphorylation will be discussed later.

ATP has special roles in energy coupling & Pi transfer. DG of phosphate hydrolysis from ATP is intermediate among examples below. ATP can thus act as a Pi donor, & ATP can be synthesized by Pi transfer, e.g., from PEP. Compound

DGo' of phosphate hydrolysis, kJ/mol

Phosphoenolpyruvate (PEP)

-

Phosphocreatine

-

Pyrophosphate

-

ATP (to ADP)

-

Glucose-6-phosphate

-

Glycerol-3-phosphate

-

O

Some other “high energy” bonds:

Coenzyme A-SH + HO

C

R

O Coenzyme A-S

C

R

+ H2O

A thioester forms between a carboxylic acid & a thiol (SH), e.g., the thiol of coenzyme A (abbreviated CoA-SH). Thioesters are ~ linkages. In contrast to phosphate esters, thioesters have a large negative DG of hydrolysis.

O C CH3 Coenzyme A-SH + HO acetic acid O Coenzyme A-S

C

CH3 + H2O

acetyl-CoA

The thiol of coenzyme A can react with a carboxyl group of acetic acid (yielding acetyl-CoA) or a fatty acid (yielding fatty acyl-CoA). The spontaneity of thioester cleavage is essential to the role of coenzyme A as an acyl group carrier. Like ATP, CoA has a high group transfer potential.

SH CH2

Coenzyme A includes b-mercaptoethylamine, in amide linkage to the carboxyl group of the B vitamin pantothenate. The hydroxyl of pantothenate is in ester linkage to a phosphate of ADP-3'-phosphate. The functional group is the thiol (SH) of b-mercaptoethylamine.

b-mercaptoethylamine

CH2 NH C

O

CH2

pantothenate

CH2 NH C

NH2 O

HO

C

H

H3C

C

CH3 O

H2C

O

N

N

P O-

O O

P

N

N O

CH2

O-

O

H

H

O

H OH

H

ADP-3'-phosphate -

Coenzyme A

O

P O

O-

3',5'-Cyclic AMP (cAMP), is used by cells as a transient signal. Adenylate Cyclase catalyzes cAMP synthesis: ATP  cAMP + PPi. The reaction is highly spontaneous due to the production of PPi, which spontaneously hydrolyzes.

NH2

cAMP

N

N

N

N H2 5' C 4' O

O H

H 3' P O

H

1'

2' H

OH Phosphodiesterase catalyzes O hydrolytic cleavage of one Pi ester O(red), converting cAMP  5'-AMP. This is a highly spontaneous reaction, because cAMP is sterically constrained by having a phosphate with ester links to 2 hydroxyls of the same ribose. The lability of cAMP to hydrolysis makes it an excellent transient signal.

List compounds exemplifying the following roles of "high energy" bonds:  Energy transfer or storage ATP, PPi, polyphosphate, phosphocreatine  Group transfer ATP, Coenzyme A  Transient signal cyclic AMP

Kinetics vs Thermodynamics: A high activation energy barrier usually causes hydrolysis of a “high energy” bond to be very slow in the absence of an enzyme catalyst. This kinetic stability is essential to the role of ATP and other compounds with ~ bonds. If ATP would rapidly hydrolyze in the absence of a catalyst, it could not serve its important roles in energy metabolism and phosphate transfer. Phosphate is removed from ATP only when the reaction is coupled via enzyme catalysis to some other reaction useful to the cell, such as transport of an ion, phosphorylation of glucose, or regulation of an enzyme by phosphorylation of a serine residue.

Oxidation & reduction

Oxidation of an iron atom involves loss of an electron (to an acceptor): Fe++ (reduced)  Fe+++ (oxidized) + eSince electrons in a C-O bond are associated more with O, increased oxidation of a C atom means increased number of C-O bonds. H H

C

H H H

C

OH H

H

O

O

O

C

C

C

H H

H

OH O

Increasing oxidation of carbon

Oxidation of carbon is spontaneous (energy-yielding). Two important e- carriers in metabolism: NAD+ & FAD.

NAD+, Nicotinamide Adenine Dinucleotide, is an electron acceptor in catabolic pathways. The nicotinamide ring, derived from the vitamin niacin, accepts 2 e- & 1 H+ (a hydride) in going to the reduced state, NADH.

NADP+/NADPH is similar except for Pi. NADPH is e- donor in synthetic pathways.

Nicotinamide Adenine Dinucleotide

H

C NH 2

O -

O

P

O

+ N

CH 2

O

H

H OH

OH NH 2

O

N

N

O

P

nicotinamide

H

H

-

O

O

CH 2

O

H

N O

adenine

H

esterified to Pi in NADP+

H

H OH

N

OH

NAD+/NADH H

O

H

H

C

C NH2

+ N

O

 2 e- + H+

NH2 N

R

R

NAD+

NADH

The electron transfer reaction may be summarized as : NAD+ + 2e- + H+  NADH. It may also be written as: NAD+ + 2e- + 2H+  NADH + H+

dimethylisoalloxazine

O

H C

C

N

O -

H3C

C

C

C

NH

H3C

C

C

C

C

C H

N

H C

+

2e +2H O

N

H N

H3C

C

C

C

NH

H3C

C

C

C

C

C H

CH2

FAD

N

O

N H

CH2

HC

OH

HC

OH

HC

OH O

H2C

C

O

P O-

Adenine

O O

P O-

O

Ribose

FADH2

HC

OH

HC

OH

HC

OH O

H2C

O

P O-

Adenine

O O

P

O

O-

FAD (Flavin Adenine Dinucleotide), derived from the vitamin riboflavin, functions as an e- acceptor. The dimethylisoalloxazine ring undergoes reduction/oxidation. FAD accepts 2 e- + 2 H+ in going to its reduced state: FAD + 2 e- + 2 H+  FADH2

Ribose

 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.