01: Introduction to Cellular MetabolismPrinciples of Metabolic Regulation

Section Objectives

• Define: metabolism (anabolism, catabolism)

• Describe the process of regulatory feedback

• Explain the role of negative feedback

• Describe the control mechanisms that regulate metabolism

• Describe the role of diet and nutrition in the production of energy

• Describe the role of ATP and Coenzyme A as carriers

• Describe the biochemical role of redox cofactors

  • Niacinamides

  • Flavins

Life Needs Energy

• Recall that living organisms are built of complex structures.

• Building complex structures that are low in entropy is only possible when energy is spent in the process.

• The ultimate source of this energy on Earth is sunlight

Metabolism Is the Sum of All Chemical Reactions in the Cell

Metabolism

• Definition: Primary metabolism is a collection of reactions responsible for

  • the generation of energy for the cells

  • the use of this energy, along with simple organic precursor molecules, to make more complicated molecules for the cell

• Anabolism: subset of those reactions that lead to the synthesis of complex molecules from simple precursors

  • in the definition of primary metabolism

• Catabolism: subset of those reactions that lead to the breakdown of energy- yielding molecules

  • in the definition of primary metabolism

• Metabolism is an orderly progression of chemical transformations

  • Each reaction is linked with another

• Pathway: The set of linked reactions from precursor to final product, along with enzymes, cofactors, and regulatory factors

  • Enzyme + cofactor = Holoenzyme

  • Not all enzymes need cofactors

• Pathways require some energy input

• Pathways are tightly regulated and coordinated with other pathways

• Pathways can be

  • Linear

  • Branched

  • Circular

Regulatory Feedback

• Feedback: When a portion of the output of a system (or a process) returns as input for that system (or process), it is called feedback

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Negative Feedback

• Negative feedback is more common in physiology and biochemistry, than positive feedback

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• Important features of negative feedback are:

  • The first unique step of a pathway is regulated

    • Committed step; no going back

    • End product is an inhibitor of the process/pathway

  • Intermediate products can also inhibit enzyme(s)

    • Product A on E1

    • Product B on E1 and E2

    • or Product C on E1, E2, and E3

      • The end product C may or may not resemble the precursor (or substrate) for the enzyme

• Inhibition of enzymes that control metabolic pathways results in greater efficiency

  • Inhibition is ideally reversible

• Product of the pathway binds to the enzyme and changes enzyme

  conformation

  • Competitive

  • Non-competitive

  • Allosteric inhibition

  • Reaction “stops” as a result

• Negative feedback is a regulated process

  • Only necessary reactions proceed

  • Unnecessary, wasteful loss of energy and materials is avoided

Metabolic Control Mechanisms

• Metabolism is regulated by several mechanisms

• The most important ones are:

  • Concentration

  • Compartmentalization

  • Enzyme activation and deactivation

  • Reciprocal regulation of competing pathways

Concentration

• Concentration mechanisms include the following factors:

  • Enzyme levels

    • Enzyme synthesis is controlled by genes

    • Elevated enzyme levels increases Kcat

  • Substrates

    • Availability of substrate

    • High conc. of substrate will lead to more product

    • Rate of reaction depends upon [S]

  • Cofactors

    • Availability of cofactors

    • Presence of cofactors will favor product formation

Effect of [Substrate] on Enzyme Activity

Compartmentalization

• Different or opposing pathways are placed in different cellular compartments i.e. cell organelles, or different organs (muscle v. liver)

  • Nucleus: DNA replication, synthesis of mRNA

  • Mitochondria: Krebs’ cycle, fatty acid oxidation

  • Cytosol: glycolysis, fatty acid synthesis

• The transport systems that ferries material across the membranes are also regulated

  • Affects concentration of enzymes, substrates and cofactors on either side of the membrane

  • E.g. OATP (Anion Transporter Proteins), P-glycoprotein

Control of Carbohydrate Metabolism in the Liver vs the Muscle

Enzyme Activation and Deactivation

• The activation/deactivation of enzymes directly impacts their catalytic

  activity

• Catalytic activity is influenced by substrates or inhibitors

  • Covalent modifications

  • Non-covalent modifications

• Enzymes with mutually opposing roles may co-exist in the same organelles

  • Their activity is controlled by the process of reciprocal regulation

  • E.g. phosphatases and kinases in the cytosol

    • These enzymes have opposite types of actions. Kinases load phosphate groups on molecules, while phosphatases help unload those phosphate groups.

    • Phosphate groups ... think ATP, ADP!

Phosphorylation of Enzymes Affects their Activity

• Phosphorylation is catalyzed by protein kinases.

• Dephosphorylation is catalyzed by protein phosphatases

  • or they can be spontaneous.

  • Typically, proteins are phosphorylated on the hydroxyl groups of Ser, Thr, or Tyr.

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Reciprocal Regulation

• The cell contains many enzymes for biosynthesis of molecules

  • These molecules are necessary for the cell’s function

• Some enzymes may have opposing activities

  • Compartmentalization may not always work

  • Enzyme activity carefully controlled to prevent wastage

• Control is done by reciprocal regulation

• Definition: Depending upon the immediate physiological state, one set of enzymes have to be shut off when the other set of enzymes is active.

Glycolysis vs Gluconeogenesis

• Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme.

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Major Food Components

• Food is a complex mixture of

  • Carbohydrates, Lipids (fats), Proteins (major components)

  • Vitamins and minerals (minor components)

• Carbohydrates (sugars)

  • Complex sugars (polysaccharides)

  • Simple sugars (monosaccharides and disaccharides)

• Lipids (fats)

  • Triglycerides (aka Triacylglycerols)

• Proteins

  • Amino acids

Diet and Nutrition

• The body needs energy for its various functions

• Energy is obtained from a proper diet

  • Oxidation of carbohydrates and lipids releases energy

  • Amino acids are needed to build nucleic acids and proteins

    • Enzymes!

  • Vitamins, minerals, are not synthesized by the body

    • They have to be obtained from the diet

• Water is a major component of the cell

  • Transport of molecules, ions

  • Dissolution of molecules, ions

  • Solvent medium for cellular reactions

Stages of Digestion of Food

• Polymeric foodstuff is broken down to monomeric components (Stage I)

  • Enzymes in the mouth and gut; acid in the stomach; bile salts in the intestine

• Monomeric components are converted to simple metabolic intermediates (Stage II)

  • Components are transported to the cell by the blood; conversion occurs within the cell

• Degradation of simple intermediates to CO2, NH3, water and urea (Stage III)

  • Catalyzed by enzymes

  • Energy-generation via metabolic pathways

Metabolic Pathways for Digestion

• Carbohydrates, lipids, and proteins are sources of energy

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• Metabolic pathways involved in the breakdown to release energy

• Polysaccharides are hydrolyzed to mono- and disaccharides

• Triglycerides are hydrolyzed to “free” fatty acids and glycerol

• Proteins are hydrolyzed to amino acids

Energy and ATP

• Activated group carriers reduce energy barriers

  • Makes substrates more reactive

  • Electrophilic and Nucleophilic reactions

  • Acid-base catalysis

• Enables the conversion of substrates to products

• Many are co-substrates

  • Derived from vitamins

  • E.g. Adenosine Triphosphate (ATP), Coenzyme A (CoA), S-Adenosyl Methionine (SAM)

• Pro-tip #1: Think of these molecules as good leaving groups

Adenosine Triphosphate (ATP)

• Adenosine Triphosphate

  • Adenine (Nitrogen base)

  • Ribose (pentose sugar)

  • Phosphate groups

    • 3: triphosphate (ATP)

    • 2: diphosphate (ADP)

    • 1: monophosphate (AMP)

  • Hydrolysis of phosphate bonds releases huge amounts of energy

    • 7.3 kcal/mol (30.5 kJ/mol)

CoEnzyme A

• Coenzyme A (CoA, CoASH) transfers activated acyl groups

  • The R group in carboxylic acids are acyl groups

  • “Acetyl” is a name for a specific type of acyl group

  • Acetyl CoA is an important energy molecule

    • E.g. fatty acid metabolism

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Redox Cofactors

• Metabolism involves sequential oxidation of molecules obtained from our diet

  • Oxidation-Reduction reactions: Redox

• Redox reactions involve electrons

  • Recall: OIL RIG; LEO GER

  • Electron transport chain in the mitochondria

• Helper molecules in the cell assist in redox reactions

  • Organic molecules (possess ionizable groups)

  • Metals (accept and donate electrons)

• Nicotinamide Adenine Dinucleotide (NAD+ and NADH)

• Flavin Adenine Dinucleotide (FAD and FADH2)

NAD and NADP are Common Redox Cofactors

• These are commonly called pyridine nucleotides.

• They can dissociate from the enzyme after the reaction.

• In a typical biological oxidation reaction, hydride from an alcohol is transferred to NAD+, giving NADH

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  • Nicotinamide adenine dinucleotide, NAD+, and its phosphorylated analog NADP+ undergo reduction to NADH and NADPH

Formation of NADH can be Monitored by UV-Spectrophotometry

• Measure the change of absorbance at 340 nm

• Very useful signal when studying the kinetics of NAD-dependent dehydrogenases

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Lesson in Quantum Chemistry

• Most organic molecules, including the reduced fuels, are in the singlet spin

  state.

  • All electrons are paired into electron pairs.

• Molecular oxygen is in the triplet spin state

  • Two electrons are unpaired.

• Direct electron transfer from a singlet reduced species to a triplet-oxidizing species is quantum-mechanically unfavorable.

  • This is why direct oxidation (spontaneous combustion) of biomolecules does not occur readily.

• Cofactors, such as transition metal ions and FAD, are able to catalyze consecutive single-electron transfers needed for utilization of O2.

Flavin Cofactors Allow Single Electron Transfers

• Permits the use of molecular oxygen as an ultimate electron acceptor

  • flavin-dependent oxidases

• Flavin cofactors are tightly bound to proteins.

Nicotinamide

• Co-substrate derived from niacin (Vitamin B3)

  • NADH (Nicotinamide Adenine Dinucleotide)

  • NADPH (Nicotinamide Adenine Dinucleotide Phosphate)

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• Structure of Nicotinamide

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Flavins

• Derived from riboflavin (Vitamin B2)

  • FAD (Flavin Adenine Dinucleotide)

    • FADH2 is the reduced form

  • FMN (Flavin Mono Nucleotide)

  • FMNH2 is the reduced form

• Flavins participate in redox reactions

  • 1 e- or 2 e-

  • Conjugated central ring serves as an electron sink

• Pro-tip #3: “Electron Sink” Think of it as a sink where electrons are “drained”

• Structure

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