3 Cell Metabolism

Chapter 3 Cell Metabolism - review Student Learning Outcomes:

• Describe central role of enzymes as catalysts Vast array of chemical reactions

Many enzymes are proteinsRole of NAD+/NADH coenzyme carrying electrons

• Explain how metabolic energy comes from breaking/ rejoining covalent bonds: ATP is energy currency

• Glycolysis, fermentation or aerobic respiration• What goes into reactions, what comes out

• Briefly explain biosynthesis of cell constituents (requires energy)

Fig 3.1 Energy diagrams for catalyzed and uncatalyzed reactions

Enzymes: catalysts increase rate of chemical reactions in cells (lower activation energy)• not consumed in reaction• not alter chemical equilibrium between reactants and products

Conversion of substrate (S) to product (P):

Fig. 3.1

Enzymes bind substrates to form enzyme-substrate complex (ES)• S binds to active site of enzyme.• S converted to product, released

PEESES

Fig. 3.2 Enzymatic catalysis of reaction between 2 substrates

• Biochemical reactions often 2 or more substrates.ex: peptide bond joins 2 amino acids

• Enzyme brings substrates together in proper orientation to favor transition state

• Active sites: clefts or grooves from tertiary structure• Substrates bind active site:

hydrogen bonds, ionic bonds, hydrophobic interactions.

Fig. 3.2

Fig 3.3 Models of enzyme-substrate interaction

Lock-and-key model:• substrate fits precisely active site.**Induced fit: • modifies configurations of both enzyme and substrate

Specific side chains in active site may react with substrate and form bondswith reaction intermediates

Fig. 3.3

Fig 3.4 Substrate binding by serine proteases

Ex. Chymotrypsin digests proteins by catalyzing hydrolysis of peptide bonds

Chymotrypsin digests adjacent to hydrophobic amino acids

Trypsin digests next to basic amino acids

• Nature of active site pocket determines substrate specificity of different proteases

• Active site amino acids are involved in reaction

212 PeptidePeptideOHoteinPr

Fig. 3.4

Fig 3.5 Catalytic mechanism of chymotrypsin

Chymotrypsin: Substrate binding orients peptide bond adjacent to serine in active site; catalytic reaction involves covalent join to serine.

1

2 3

The Central Role of Enzymes as Biological Catalysts

Small molecules binding in active sites assist catalysis.

Prosthetic groups: small molecules bound to proteins - critical functional roles.Ex: myoglobin and hemoglobin,

prosthetic group is heme, which binds O2. Ex. metal ions (zinc, iron)

Coenzymes: low-molecular-weight organic molecules that work with enzymes to enhance reaction rates. Ex. NAD+ works with many enzyme (carries electrons)

Fig 3.6 Role of NAD+ in oxidation–reduction reactions

Nicotinamide adenine dinucleotide (NAD+): REDOX: coenzyme carries electrons in oxidation: reduction reactions

NAD+ accepts H+ and 2 e- from one substrate →NADH. NADH donates these e- to second substrate, re-forming NAD+.

Fig. 3.6

S1 (red) + S2 (ox) -> S1 (ox) + S2 (red)

Enzymes and coenzymes

Some coenzymes are related to vitamins

Fig 3.7 Feedback inhibition

Enzyme activity is often regulated• Ex. feedback inhibition, product of pathway inhibits an enzyme

involved in its synthesis.

Fig. 3.7

Fig 3.8 Allosteric regulation

Feedback inhibition is example of allosteric regulation: enzyme activity controlled by binding of small molecules to regulatory sites on enzyme (not at active site)

• changes conformation of enzyme and alters active site.

Fig. 3.8

Fig 3.9 Protein phosphorylation

**Enzyme activity can be modified by phosphorylation: addition of phosphate can stimulate or inhibit activity of an enzyme.

Kinases addPhosphate(-OH of ser, thr, tyr)

PhosphatasesremovePhosphate

Fig. 3.9 ex. phosphorylation activates enzyme that degrades glycogen

Metabolic Energy

• A large portion of cell’s activities is devoted to obtaining energy from environment, and using energy to drive energy-requiring reactions

Many reactions in cells are energetically unfavorable, can proceed only with energy input

(especially biosynthetic reactions)

ATP and NADH provide energy and reducing material (e-) for coupled reactions

Fig 3.10 ATP as a store of energy

Adenosine 5′-triphosphate (ATP) plays central role in storing, using free energy in the cell –

Energy currency.Bonds between phosphates in ATP are high-energy bonds:

Hydrolysis is accompanied by large decrease in free energy:powers coupled reactions.

Fig. 3.10

Metabolic Energy

Hydrolysis of ATP drives energy-requiring reactions

Ex: first step in glycolysis is unfavorable (ΔG°′ = +3.3)

OHphosphateecosgluHPOecosglu 224 6

ATP hydrolysis is energy yielding: (ΔG °′ = –7.3 kcal/mol):

Combined (coupled) reaction : (ΔG°′ = -4.0) kcal/mol)

242 HPOADPOHATP

ADPphosphateecosgluATPecosglu 6

Energy-yielding reactions - coupled to ATP synthesisEnergy-requiring reactions - coupled to ATP hydrolysis.

Metabolic Energy

Ex. complete oxidative breakdown of glucose to CO2 and H2O yields large amount of free energy:

ΔG°′ = –686 kcal/mol.

OHCOOOHC 2226126 66

To harness this energy, glucose is oxidized in a series of steps coupled to ATP synthesisGlycolysis, citric acid cycle, e- transport chain (Krebs cycle), (oxidative phosphorylation)

Energy-yielding reactions - coupled to ATP synthesisEnergy-requiring reactions - coupled to ATP hydrolysis.

Metabolic Energy

Glycolysis: common to all cells; does not require O2

• Anaerobic organisms, can provide all metabolic energy (ex. E. coli, Streptococcus, yeast).

• Aerobic cells, only 1st stage in glucose degradation

Glycolysis:Breakdown of glucose -> 2 pyruvate, net gain 2 ATP

• Enzymes that catalyze reactions are regulatory points: if adequate supply of ATP, glycolysis is inhibited

Also converts 2 molecules of NAD+ to NADH:• NADH must be recycled by donating e- for other

oxidation–reduction reactions.

.

Figure 3.11 Reactions of glycolysis

Fig. 3.11

Glycolysis: 1 glucose → 2 pyruvate, net gain of 2 ATP• First part of pathway consumes energy (2 ATP)• Second part generates energy (4 ATP)

Also converts 2 molecules of NAD+ to NADH:• NAD+ is oxidizing agent that accepts e-• NADH must be recycled by donating e- for other REDOX

pyruvate

Metabolic Energy

In eukaryotic cells, glycolysis in cytosol.

NADH must be recycled, donate e- for other REDOX:• Anaerobic conditions, NADH reoxidized to NAD+ by

conversion of pyruvate to lactate or ethanol (fermentation):• Wasteful process reduces pyruvate, low ATP gain

• Aerobic conditions, NADH donates e- to electron transport chain (oxidative respiration) (lot of ATP)

• Pyruvate is transported into mitochondria, for complete oxidation (Krebs + electron transport chain)

• (citric acid cycle)

Fig 3.12 Oxidative decarboxylation of pyruvate

Pyruvate oxidative decarboxylation with coenzyme A (CoA-SH): forms acetyl CoA and more NADH.

Fig. 3.12

Fig 3.13 The citric acid cycle

Acetyl CoA enters citric acid cycle (Krebs cycle)• 2-C acetyl group + oxaloacetate (4-C) yields citrate (6-C).• 2 C of citrate are completely oxidized to CO2;

• oxaloacetate is regenerated.

Citric acid cycle:

completes oxidation of glucose to 6 CO2

Each Acetyl-CoA yields:

2 CO2, 1 GTP, 3 NADH,

1 flavin adenine dinucleotide (FADH2), (another e- carrier.

Fig. 3.13

Oxidative phosphorylation summary

Fig. 21.1 Lieberman & Marks, Basic Medical Biochemistry

Oxidative phosphorylation: electrons of NADH, FADH2 combine with O2; energy released drives synthesis of ATP.

• Passage of e- through carriers: electron transport chain, inner mitochondrial membrane of eukaryotes

(inner plasma membrane of prokaryotes)• H+ are pumped out → electrochemical gradient• H+ back in through ATP synthase makes ATP (~3/NADH)

Metabolic Energy

Glucose breakdown (O2) to CO2, H2O → ~36-38 ATP

Breakdown of other organic molecules yields energy:

• Nucleotides and polysaccharides are broken down to sugars which enter glycolytic pathway

• Amino acids are degraded via citric acid cycle.

• Fats (triacylglycerols) broken to glycerol and free fatty acids.• fatty acid joins to coenzyme A, yields fatty acyl-CoA• fatty acids degraded stepwise process, two C at a time• Yield 1 Acetyl CoA, 1 NADH, 1 FADH2 each cycle• ~130 ATPs per molecule of 16-carbon fatty acid.

Fig 3.15 Oxidation of fatty acids

Fig. 3.15

Each round of fatty acid oxidation yields one NADH, one FADH2.

Acetyl CoA enters citric acid cycle (for complete oxidation).

Net gain: ~130 ATPs per molecule of 16-carbon fatty acid

(net gain of ~38 ATPs per molecule of glucose with 6 C).

Photosynthesis brief

Photosynthesis converts energy of sunlight to usable form of chemical energy.

• ultimate source of all metabolic energy in biological systems.

• Overall equation for photosynthesis:

Process takes place in two stages: • Light reactions: sunlight energy drives synthesis of ATP

and NADPH, coupled to oxidation of H2O to O2.• Dark reactions: ATP and NADPH drive synthesis of

carbohydrates from CO2

• 18 ATP and 12 NADPH required for each glucose

2612622 666 OOHCOHCO light

Photosynthesis

Photosynthetic pigments absorb photons of light; shifts electrons into higher energy orbitals, convert energy from sunlight to chemical energy in ATP, and also NADPH

In eukaryotic cells, reactions occur in chloroplasts In prokaryotic cells, reactions occur on plasma membraneChlorophylls major pigments; other pigments absorb different wavelengths of light

Fig. 3.16

Fig 3.18 The Calvin cycle

Light reactions: energy from light converts H2O to O2.High-energy electrons enter electron transport chain: transfer

through series of carriers is coupled to synthesis of ATP.

Dark reactions: ATP and NADPH drive synthesis of carbohydrates from CO2 and H2O.

• One molecule of CO2 added each cycle of reactions, Calvin cycle, that

forms carbohydrates.

Fig. 3.17,18

The Biosynthesis of Cell Constituents

Biosynthesis of cell constuents**Energy derived from breakdown of organic

molecules (catabolism) drives synthesis of other components of cell.

Biosynthetic (anabolic) pathways use ATP and reducing power (usually NADPH) to produce new organic compounds.

Animal cells: glucose synthesis (gluconeogenesis) usually starts with lactate (from anaerobic glycolysis), amino acids (breakdown of proteins), or glycerol (breakdown of lipids):

• pyruvate is converted to glucose: • not just reversal of glycolysis; requires more energy for

biosynthesis of glucose than get from breakdown

Fig 3.20 Synthesis of polysaccharides

Glucose is stored as starch and glycogen.Synthesis of polysaccharides requires energy.• Dehydration reaction joining sugars is unfavorable, couples to energy-yielding reaction; nucleotide sugar intermediates.•Glucose phosphorylated, reacts with UTP → UDP-glucose.

•UDP-glucose (activated intermediate) donates glucose to growing polysaccharide chain.

Fig. 3.20

The Biosynthesis of Cell Constituents

Lipids are important energy storage molecules and major constituent of cell membranes.

Fatty acids are synthesized from acetyl CoA, (from the breakdown of carbohydrates), in reactions that resemble reverse of fatty acid oxidation.

• Requires ATP, NADPH

Fig 3.22 Biosynthesis of amino acids

Amino acids are derived from diet (some are essential), or formed from citric acid intermediates

• NH3 is incorporated during synthesis of Glu and Gln• These amino acids donate NH3 to form other amino acids, (derived from intermediates in glycolysis, citric acid cycle)• Many bacteria and plants can synthesize all 20 aa

Fig. 3.22

Molecular Medicine 3.1 Phenylketonuria: Abnormal metabolism of phenylalanine in patients with phenylketonuria

Errors in amino acid metabolism can have large impacts

Ex. phenylketonuria is deficiency of phenylalanine hydroxylase, which converts phenylalanine to tyrosine.

Phenylalanine and metabolites accumulate and cause mental retardation.

Newborns tested, special diet

The Biosynthesis of Cell Constituents

Synthesis of proteins: Amino acids are incorporated into proteins in order specified by

nucleotide bases in genemRNA is template for protein synthesis on ribosome• Each amino acid is attached to specific transfer RNA (tRNA) molecule in

reaction coupled to ATP hydrolysis (charging on 3’ position)• Aminoacyl-tRNAs align on mRNA bound on ribosome;• Peptide chain joins to new tRNA-aa, coupled to hydrolysis of GTP

Fig. 3.23

Fig 3.24 Biosynthesis of purine and pyrimidine nucleotides

Nucleotides can be synthesized from carbohydrates and amino acids, or reused following nucleic acid breakdown.

Ribose-5-phosphate is starting point for nucleotide synthesis. Different pathways for synthesis of purine and pyrimidine.

Ribonucleotides

are converted to

deoxyribonucleotides,

building blocks of DNA

Fig. 3.24

Fig 3.25 Synthesis of polynucleotides

Nucleic acid synthesis requires energy;NTPs are activated precursors.

Fig. 3.25

Review

Review Questions:

1. Binding pocket of trypsin contains Asp residue. How would changing this aa to Lys affect enzyme’s activity?

4. Many biochemical reactions (synthesis of macromolecules) are energetically unfavorable under physiological conditions. How does cell carry out these reactions?

8. Yeast can grow anaerobic or aerobic. For every molecule of glucose consumed, compare number of ATP generated in anaerobic versus aerobic conditions.

10. How do organisms growing under anaerobic conditions regenerate NAD+ from NADH produced during glycolysis?