Biochemistry (II)

Introduction to metabolism & common reactions

• This course is aimed for you to be able to understand the major biochemicals processes that occur in
biology
• Biological energy generation & storage
• Biochemical synthesis and degradation of biomolecules
(sugars, lipids, amino acids, nucleic acids)

Reference material
Biochemistry 4th edition, Mathews, Van Holde, Appling, Anthony-Cahill. Pearson ISBN:978-0-13-800464-4 1
Lehninger Principles of Biochemistry 4th edition, David L. Nelson, Michael M. Cox. W. H. Freeman ISBN:978-0716743392
 Metabolism

Metabolism is the study of chemical reactions and their regulations inside a cell…

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• By studying metabolism, we can:

• Understand human diet so we

eat healthier
• Understand human diseases so
we can try to treat them
• Make food & beverages
• Discover new enzymes and
metabolic pathways for
industrial applications
• Design cell factories so that we
can produce expensive
compound cheaper
• Environmental production
• Understand how evolution
comes about
• Many many more…

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Figure from Nakayama et al. 2008. DNA Research 15, 185-199
 Spring of 2024
Week Topic
1 Introduction & Common reactions
2 Carbohydrate Metabolism: glycolysis reactions and mechanisms
3 Carbohydrate Metabolism: Gluconeogenesis and fermentation
Carbohydrate Metabolism: Pentose phosphate pathway and other
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carbohydrates
5 Pyruvate dehydrogenase: synthesis of acetyl-CoA
6 TCA Cycle and Glyoxylate Shunt
7 National Holliday: No class
8 Electron transport chain & oxidative phosphorylation
9 4/19: midterm exam
10 Photosynthesis light reaction
11 Photosynthesis dark reaction
12 Lipid Metabolism I: Fatty acid degradation
13 Lipid Metabolism I: Fatty acid biosynthesis
14 Polyketides & steroids
Nitrogen in biochemistry: nitrogen assimilation & urea cycle
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5/31: Metabolic pathway compilation assignment due
16 6/7: Final Exam 4
 Grading

Semester grade breakdown

• Midterm Exam (45%)

• Final Exam (45%)

• Assignment: Metabolic pathway compilation (10%)

Regrading policy:
• Mid-term exams will be returned to students. Any regrades necessary must be submitted directly
after the return of the exam. No exam regrades will be made past that time.
 Catabolism vs. Anabolism

• Catabolism: reactions that break down

nutrients and collect released energy and
reducing power
• Catabolic pathways are convergent

• Anabolism: reactions that synthesize needed

compounds, using stored energy and reducing
power
• Anabolic pathways are divergent

• Metabolites: chemicals/compounds that are

intermediates and/or products of metabolism.
These are usually referring to small molecules.

• Pathway: a set of reactions that transform a

compound or a class of compounds in
metabolism.
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Converging Catabolism vs. Diverging Anabolism

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 Metabolic currency ($$$)
• Metabolic intermediates serves as “currency” to convert one type of molecule to another.

NADH Electron carriers

Complex metabolites
NADPH Participates in
FADH2 Oxidation and
ADP + Pi reductions

NADPH ATP = Energy carrier

Used to increase
thermodynamic favorability
DEGRADATION BIOSYNTHESIS of reaction

NADPH

ATP

Simple metabolites

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Today’s topics:

1. ATP & other high energy phosphates

2. Oxidation & reduction (NADH, NADPH, FADH2)

3. Common functional groups and reactions in biochemistry

4. General pathway regulations

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ATP & High energy phosphate donors
• Why ATP hydrolysis is exergonic
• How ATP aids in metabolic reactions
• Make ATP by using other high energy phosphate carriers
• Examples of why high energy phosphate donors release energy upon hydrolysis

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 The adenylates (ATP, ADP, AMP) are the primary energy currency
• The fundamental biological role of ATP as an energy-coupling compound is to convert thermodynamically unfavorable
processes into favorable processes.
• Activated intermediates, such as ATP, allow reactions to occur under physiologically relevant concentrations of metabolic
intermediates.
• Reduced charge repulsion in products
• Better resonance stabilization of products
• More favored solvation of products
 ΔG'° is -30.5 kJ/mol0

Resonance makes Phosphate (Pi or PO4)very stable

ATP + H2O → ADP + Pi is VERY favorable reaction

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The ΔG of ATP hydrolysis is large and negative

• Although hydrolysis of ATP is thermodynamically

favorable, kinetic barrier is large (high activation
energy) without an enzyme.

• Therefore, it does not rapidly degrade and is stable

inside of the cell

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ATP is used to DRIVE thermodynamically unfavorable reactions

Pi

ATP ADP

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ΔG'° values of phosphate hydrolysis reflect ‘phosphoryl transfer potential’ (ptp)

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 Other ‘high-energy phosphate’ compounds have great stabilization of hydrolysis products

Reduced charge repulsion and enol-keto tautomerization:

ΔGʹ° = - 61.9 kJ/mol

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 Other ‘high-energy phosphate’ compounds have great stabilization of hydrolysis products

Reduced charge repulsion and resonance stabilization:

ΔGʹ° = - 49.3 kJ/mol

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 Thioesters also serve as energy currencies
Resonance of carboxylic acid stabilizes the product
Some bacteria can use acetyl-
CoA to produce acetate + ATP
Pi 9.8 kJ/mol
CoA-SH
phosphate
acetyltransferase

Acetyl-Phosphate
Acetate
kinase
ADP
ATP -13 kJ/mol
Similar ΔG'° of
hydrolysis as ATP

ΔGʹ° = - 31.4 kJ/mol

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Oxidation & Reduction
• Reducing cofactors used in biochemistry
• Reaction mechanism associated with electron transfer
• NAD is primarily used for catabolism (energy generation)
• NADP is primarily used for anabolism (biosynthesis)

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Simple oxidation and reduction (Redox) in common context of metabolism:

Reduced Oxidized

2e- 2e- 2e-

CO2

methanol formaldehyde Formic acid

(or formate)

oxidation

Reduction

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 Redox energy currencies transfer reducing power (ex: NAD and NADP)

• 2 electron, 1 proton carriers

• cosubstrates: diffuse between different enzymes

• NAD: primarily used in catabolism

• NADP: primarily used in anabolism

NAD: X = H
NADP: X = PO32-
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NAD+ accepts a “hydride” to become NADH

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 Cells use NADH as a reducing agent to reduce metabolites
Oxidation/reduction: NADH dependent Lactate dehydrogenase

Hydride transfer (H:-) from NADH to Pyruvate, forming lactate

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 Oxidation/Reduction reactions (Redox)
• Energy production in most cells involves the oxidation of fuel molecules such as glucose and fats.

• Oxidation-reduction, or redox, chemistry is the core of metabolism.

• Redox reactions involve reversible electron transfer from a donor (the reductant) to an acceptor (the oxidant).

• In below example, because the alcohol has lost a pair of electrons and two hydrogen atoms (essentially H2),
• this type of oxidation is called dehydrogenation, and enzymes that catalyze this reaction are called
dehydrogenase. NADH is called reducing cofactor, reducing equivalent, or sometimes reducing power

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 NADH vs. NADPH
• NAD+ is the cofactor for most enzymes that act in the direction of substrate oxidation
(dehydrogenases).

• NADPH usually functions as a cofactor for reductases, enzymes that catalyze substrate
reduction.

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 FAD and FMN are other redox currencies

FAD: Flavin adenosine dinucleotide FMN: Flavin mononucleotide

Riboflavin
(Vitamin B2)

Adenosine

• Usually prosthetic groups: tightly bound to enzyme

• Can transfer 1 or 2 electrons (plus 1 or 2 protons)

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FAD and FMN are other redox currencies

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Common functional groups & reactions
• Name of common functional groups
• Common nucleophiles and electrophiles in biochemistry
• Examples of important carbonyl chemistries
• Importance of α-carbon deprotonation

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 Common functional groups in biochemistry
Functional groups are groups of atoms added to carbon skeleton that have specific properties.

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Common functional groups in biochemistry

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Common biochemical nucleophiles & electrophiles

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 Carbonyl molecules

• Carbonyl chemistry account for a large percentage of biochemical reactions because the
vast majority of biological molecules contain them.

• Most of the chemistry of carbonyl groups involves nucleophiles (usually denoted as Nu:)
and electrophiles.

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 Oxyanion mechanism
• Oxyanion chemistries occur frequently to reactions involving carbonyls.

• A nucleophile attacks the carbonyl center (electrophile), generating a tetrahedral oxyanion

intermediate.

• In the case of a substitution reaction, when negative charge on oxygen comes back down, the –OR group
leaves the molecule, completing the reaction.

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Oxyanion chemistries

Reduction is used throughout metabolism

Variant of this reaction is used

in saccharide bond formation

Frequently observed in enzyme

catalytic mechanism

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 α-carbon of carbonyl has slightly acidic proton (α-hydrogen)

• α-hydrogen is weakly acidic proton (compared to other C-H which is not acidic) that can
be pulled off by a base.

• It is weakly acidic because its conjugate base is stabilized via enolate resonance.

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 Enolate intermediate enables Aldol and Claisen condensation

Forming Carbon – Carbon Bonds

Carbonyl condensation reactions:

• These reactions are initiated by

deprotonation of the weakly acidic
α-hydrogen to give a resonance-
stabilized enolate ion (top).

• Aldol condensation (left): the

enolate adds to an aldehyde or
ketone, yielding a β-hydroxy
product.

• Claisen condensation (right side):

the enolate adds to an ester (can
also be CoA-thioester), yielding a
β-keto product.

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Elimination reaction: dehydration of β-hydroxyl group

• Dehydration of β-hydroxyl group is a common

mechanism used in:
• fatty acid biosynthesis
• TCA cycle
• Amino acid biosynthesis

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General pathway regulations
• Pathway regulations (controls) are usually at enzymes that catalyze large negative ΔG steps (irreversible steps)
• Feedback controls are frequently observed in pathways
• Enzymes can be covalently modified (irreversible inhibition)
• Covalent modification can be removed using another enzyme
• Generic way to study pathway by generating mutants with inhibited enzymes

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 Enzyme regulation and control
• Pathway regulations (controls) are typically placed on enzymatic steps with large negative ΔG (representing
irreversible steps)
• These regulations can occur at various levels (transcriptional, translational, protein and substrate levels such as
allosteric inhibitions)

Both pathways are favorable, so cell can

A reaction with A reaction with control when to turn on which pathway…
small or zero ΔG: large negative ΔG:

Can’t control Control whether or

anything… not reaction occurs
is meaningful
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 Enzyme regulation and control
• While cells have transcriptional and translational controls over gene expression and protein translation, it is also
necessary to have protein level regulation to ensure proper balance of cellular resources.

• Feedback controls:
• Negative feedback control: metabolites downstream of a pathway can inhibit upstream enzymes of the
same metabolic pathway
• Activation feedback control: product of a pathway may activate some other pathways

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 How are downstream products able to control upstream enzymes?
• Allosteric regulations – interactions between enzyme and a molecule that induces enzyme to undergo conformational
change.
• Conformational change may increase enzyme activity or decrease.
• Example: Aspartate carbamoyltransferase (catalyzing the committed step for pyrimidine biosynthesis)

pyrimidines

CTP
ATP

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 Enzyme regulations: covalent modifications
Covalent modifications are ways to “irreversibly” inhibit an enzyme
This covalent modification can be removed by another enzyme
(for example, kinase for phosphorylation & phosphatase for de-phosphorylation)

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 Reversible covalent modification by kinases/phosphatases:
• Irreversible inhibition can be cancelled by enzymes that remove the modification

• Ex. Phosphorylation by a kinase can be removed by phosphatase

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 How do we know enzyme’s participation in a pathway
• By inactivating individual enzymes through either mutations or enzyme inhibitors can help identify the metabolic roles of
enzymes.

• The steps of a hypothetical metabolic pathway are identified by analysis of mutants defective in individual steps of the
pathway.

A Enz 1
B Enz 2
C Enz 3
D (required for growth)

Enzyme mutated or Cannot grow Metabolite that

inhibited unless add will accumulate

Enz 1 B or C or D A

Enz 2 C or D B

Enz 3 D C

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