Introduction to Metabolism

Metabolism(The Acquisition and Utilization of Free Energy)

Catabolism: exergonic oxidationAnabolism: endergonic processes

Endergonic Processes

• Mechanical Work

• Active Transport

• Biosynthesis

Anabolism and Catabolism

exergonic

endergonic

Roles of ATP and NADP+ in Metabolism

ATP

Kinetic Stabilityof

Phosphoanhydride Bonds

ATP

Adenosine

Ribose

Triphosphate

Hydrolysis of ATP

Phosphate Compounds

Roles of ATP(Coupled Reactions)

∆Go’

(kJ/mol) ----------

Fructose-6-P + Pi ——> Fructose-1,6-bisP + H2O +13.3 ATP + H2O ——> ADP + Pi -30.5-------------------------------------------------------------------------------Fructose-6-P + ATP ——> Fructose-1,6-bisP + ADP -17.2

Roles of ATP

• Early stages of nutrient breakdown

Glucose + ATP ——> Glucose-6-P + ADP

• Interconverson of nucleoside triphosphtes

NDP + ATP ——> NTP + ADP

Nucleoside Diphosphate Kinase

• Physiological processes

– Muscle contraction

– Active transport

Roles of ATP

• Additional phosphoanhydride cleavages in highly endergonic reactons

(NMP)n + NTP ——> (NMP)n+1 + PPi

PPi + H2O ——> 2 Pi

Pyrophosphatase

Sources of ATP

Phototrophs: photosynthesis

Chemotrophs: oxidation of organic compounds (e.g. carbohydrates, lipids, and proteins)

Formation of ATP

• Adenylate Kinase reaction 2 ADP ——> AMP + ATP

• Substrate-level phosphorylation X–P + ADP ——> X–H + ATP

• Oxidative phosphorylation

• Photophosphorylation

Substrate-Level Phosphorylation

Glyceraldehyde-3-PDehydrogenase

CHO

CHOH

CH2OPO3=

COOP

CHOH

CH2OP

1,3–Bisphosphoglycerate(1,3BPG)

+ NADH + H++ NAD+ + Pi

Glyceraldehyde-3-P(GA3P)

GA3P + NAD+ + H2O ——> 3PGA + NADH + H+ ² G°’ = –43.1 kJ/mol

3PGA + Pi ——> 1,3BPG + H2O ² G°’ = +49.3 kJ/mol

GA3P + NAD+ + Pi ——> 1,3BPG + NADH + H+ ² G°’ = +6.2 kJ/mol

COOP

CHOH

CH2OP

COOH

CHOH

CH2OP

3–Phosphoglycerate

+ ATP

1,3 Bisphosphoglycerate

+ ADP

PhosphoglycerateKinase

Oxidative Phosphorylation

Photophosphorylation

Source of NAD(P)+, and other cofactors

Source of NAD(P)+, and other cofactors

NADP+

Nicotinamide Adenine Dinucleotide (Phosphate)

N

OCH2

OH OH

O

OCH2

OH OPO3=

AOP

O

O–

P

O

O–

O

CNH2

O

Nucleotide

Nucleotide

Figure 14-1

Niacin

Figure 14-11

Reduction of NAD+ or NADP+ to NADH or NADPH

Metabolic Pathways

A ——> B ——> C ——> D ——> E

Metabolites

Enzymes

Metabolic

Map

Figure 14-3

Overview of

Catabolism

Properties of Metabolic Pathways

• Separate Anabolic and Catabolic Pathways

• Steady-State

• Irreversible (overall): reversibility of individual steps

• First Committed (Exergonic) Step: others close to equilibrium

• Compartmentation (organelles & tissues): isoenzymes and transport

• Regulation (usually first committed step): often rate-limiting

Potential Futile Cycles(Regulation)

CentralMetabolite Product/ Nutrient

Biosynthesis

Catabolism

Steady State

Input OutputA A B B

Steady-State

Thermodynamics of individual stepsA B

Go’ = -RTlnKeq

Not standard conditions or at equilibrium: G = Go’+RTln([B]/[A])

Three Physiological Conditions:Go’<<<<<<0 : G always negative

Example: ATP hydrolysisGo’>0 : near equilibrium, reversible,

direction depends on actual [B]/[A]Example: Most reactions

Go’>>>>>>0 : G always positive, must be coupledExample: Phosphorylation of Glucose

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Go’>0

Regulation of Metabolic Pathways

Specific Controls

General Controls

Specific Controls

• Control of Enzyme Amount

– Constitutive Enzymes

– Inducible Enzymes

– Repressible Enzymes

• Control of Enzyme Activity

– Regulatory Enzymes

– Effectors (Ligands)

General Controls(Integration of Cellular or Organism

Functions)

• Internal Effectors

– Catabolite Repression

– Energy Charge

– Reduction Potential

• External Effectors (e.g. hormones)

Significance: Efficiency and Flexibility!

Types of Reactions

Group Transfer Reactions

Y: + A—X Y—A + X:

Phosphoryl Group Transfer

O

CH2OH

OH

OH

OHHO

O

CH2OPO3=

OH

OH

OHHO

- D- glucose–6–P(G6P)

- D- glucose(Glc)

ATP ADP

Mg2+

Elimination Reactions

R C C R'

H

H

OH

H

C CR

H

H

R'+ H2O

Isomerization Reactions(Intramolecular Hydrogen Shifts)

H C

CH O

O H

R

Aldose

C

CH O

O H

R

BH + C

CH O

O

R

H

H C

H

O

C O

R

H

Ketose

B: +

BH

B:

Making C-C Bonds

C

H2C

COOH

COOHCH3 C S

O

CoAO

H2C

C

H2C

COOH

COOH

COOH

HO

Acetyl–SCoAOxaloacetate

Citrate

+CitrateSynthase

CoASH

Note: thioester

Breaking C-C Bonds

CH2OPO32–

C

C

C

C

CH2OPO32–

O

HHO

OHH

OHH

CHO

CHOH

CH2OPO3=

CH2OH

C O

–D–fructose- 1,6- bisphosphate(FBP)

Glyceraldehyde–3–P[GA3P]

+

Dihydroxyacetone–P [DHAP]

Aldolase

CH2OPO3=

Oxidation-Reduction Reactions

SH2 + NAD+ + H2O ——> S + NADH + H3O+

SH2: Reduced Substrate

S: Oxidized Product

NAD+: Electron Acceptor

FAD: Electron Acceptor

Figure 14-11

Reduction of NAD+ to NADH

Figure 14-12

Flavin Adenine Dinucleotide (FAD)

Figure 14-13 part 1

Reduction of FAD to FADH2

Figure 14-13 part 2

Reduction of FAD to FADH2

One Electron Oxidation-Reduction Reactions

Fe3+ + Cu+ Fe2+ + Cu2+

ElectronAcceptor

ElectronDonor

Half-Reactions

Fe3+ + e– Fe2+

Cu2+ + e–Cu+ (oxidation)

(reduction)

OxidationInvolves (e- of H:-)Loss

ReductionInvolves (e- of H:-)Gain

Alcohol Dehydrogenase(Oxidation-Reduction Reaction)

H

C

C

H

O

H

H

H

H

N

R

C NH2

O

N

R

C NH2

OH H

C

CH3

OH

++

Ethanol

++

AlcoholDehydrogenase

H+

NADHNAD+

H+

H:–

Experimental Approaches

to Metabolism

Features of Metabolic Pathways

A ——> B ——> C ——> D ——> E

(1)Sequences and Energetics

(2) Enzymes and Mechanisms

(3) Control Mechanisms (Regulation)

(4) Compartmentation

Elucidation of Metabolic Pathways

A ——> B ——> C ——> D ——> EMetabolic Inhibitors:

accumulation of intermediates

Biochemical Genetics: mutants

Pathway Labeling: isotopes

Metabolic Inhibitors(Accumulation of Intermediates)

(e.g. Glycolysis)

Fluoride: (2-phosphoglycerate and consequently 3–phosphoglycerate)

Biochemical Genetics(Mutants)

Natural Genetic DefectsManipulation of Microorganisms

Accumulation of Intermediates

Growth Requirements (auxotrophic mutants)

A ——> B ——> C ——> D ——> E

Pathway Labeling

A* ——> B* ——> C*

Stable Isotopes

Radioisotopes

Detection of Isotopes

• Stable Isotopes– Mass Spectrometry– NMR

• Radioisotopes– Proportional Counting (Geiger

Counter)– Liquid Scintillation Counting– Autoradiography

Quantify Differential Expression

Condition 1 Condition 2

Sample Prep Sample Prep

Mix samples and detect

Quantify Differences

Control of Expression

• Transcription:– Microarray

• Proteomics– 2D-SDS-PAGE– Isotope Coded Affinity Tag

ICAT Chemistry

IAM

IAM

Biotin

Biotin

ReactiveGroup

(specific forcysteines)

AffinityTag

Isotopecode

(D or 13C)

LIGHT

HEAVY

ICAT = Isotope Coded Affinity Tag

Same behavior

chemically, but different

in mass.