Biochemistry

Amino Acids Found in Proteins

Amino Acids: A molecule with 4 groups attached to a central (α) carbon: an amino group, a carboxylic acid group, a hydrogen atom, and an R Group. The R Group determines function of that amino acid.

Stereochemistry: The stereochemistry of the α-carbon is L for all chiral amino acids in eukaryotes. (carbohydrates are D-config). All chiral amino acids except cysteine have (S) configuration and all amino acids are chiral except for Glycine.

Hydrophobic & Hydrophilic: Amino acids with long alkyl chains are hydrophobic. Those with charges are hydrophilic. All others fall in somewhere in between.

Acid-Base Chemistry of Amino Acids

Amphoteric: Amino acids can act as a base or an acid.

pKa: The pH at which half of the species is deprotonated; [HA] = [A-].

pH: ↓pH ⇒ amino acid is fully protonated
pH ≈ pI ⇒ amino acid is a neutral zwitterion
↑pH ⇒ amino acid is fully deprotonated

Isoelectric Point: (pI) The pH at which an amino acid is in zwitterion form; the charges cancel out to make a neutral molecule.

pKa1 = carboxyl grp
pKa2 = amine grp
pKa3 = side chain

Titration:
Midpoint: pH = pKa
Equivalence Point: pH = pI

Peptide Bond Formation and Hydrolysis

Terminology:
Dipeptide: 2 residues
Tripeptide: 3 residues
Oligopeptides: Less than 20 residues
Polypeptides: Greater than 20 residues

Formation: Forming a peptide bond is a dehydration reaction. The nucleophilic amino group of one amino acid attacks the electrophilic carbonyl group of another amino acid.

Amide Bonds: The C-N bond of a peptide bond. Rigid due to resonance.

Breaking: Breaking a peptide bond is a hydrolysis reaction.

1° and 2° Protein Structure

1° Structure: Linear sequence of amino acids in a peptide. Stabilized by peptide bonds. The AA sequence is written N-terminus to C-terminus. N-terminus is POSITIVELY charged due to –NH3+.

2° Structure: The local structure of neighboring amino acids. Is stabilized by hydrogen bonding between amino groups and nonadjacent carboxyl groups.

α-helices: A common 2° structure. Clockwise coils around a central axis.

β-pleated sheets: A common 2° structure. Rippled strands that can be parallel or antiparallel.

Proline: Can interrupt 2° structure because of its rigid cyclic structure.

Primary Protein Structure: Sequence of a chain of amino acids

Secondary Protein Structure: Local folding of polypeptide chain into helices or sheets.

Tertiary Protein Structure: Three-dimensional folding pattern of a protein due to side chain interactions.

Quaternary Protein Structure: Protein consisting of more than one amino acid chain.

Note: Denaturing is when a protein (or nucleic acid) loses its 4°, 3°, and 2° structures due to breaking non-covalent interactions such as H-bonds, hydrophobic interactions, and dipole-dipole interactions.

3° and 4° Protein Structure

3° Structure: 3-D shape of a single polypeptide chain, and is stabilized by hydrophobic interactions, acid-base interactions, H-bonds, and disulfide bonds.

Hydrophobic Interactions: Push hydrophobic R groups to the interior of a protein, which increases entropy of the surrounding water molecules and creates a negative Gibbs free energy.

Disulfide Bonds: Occur when two cysteine molecules are oxidized and create a covalent bond between their thiol groups. This forms cystine.

4° Structure: The interaction between peptides in proteins that contain multiple subunits.

Conjugated Proteins: Proteins with covalently attached molecules.

Prosthetic Group: The attached molecule in a conjugated protein. Can be a metal ion, vitamin, lipid, carbohydrate, or nucleic acid.

Denaturation: The loss of 3-D structure. Caused by heat or solute concentration.

Enzymes

Enzymes as Biological Catalysts

Enzymes: Biological catalysts that are unchanged by the reactions they catalyze & are reusable. Enzymes DO NOT alter the ΔG or ΔH, nor the final equilibrium position. They only change the rate of reaction by altering the mechanism. Catalyze both the FORWARD & REVERSE reactions.

Exergonic Rxns: Release energy; ΔG is negative.
Endergonic Rxns: Require energy; ΔG is positive.
Oxidoreductases: REDOX reactions that involve the transfer of e⁻.
Transferases: Move a functional group from one molecule to another.
Hydrolases: Catalyze cleavage with the addition of H₂O.
Lyases: Catalyze cleavage without the addition of H₂O and without the transfer of e⁻. The reverse reaction (synthesis) is often more important biologically.
Isomerases: Catalyze the interconversion of isomers, including both constitutional isomers and stereoisomers.
Ligases: Join two large biomolecules, often of the same type.
Lipases: Catalyze the hydrolysis of fats. Dietary fats are broken down into fatty acids and glycerol or other alcohols.
Kinases: ADD a phosphate group. A type of transferase.
Phosphatases: REMOVE a phosphate group. A type of transferase.
Phosphorylases: Introduces a phosphate group into an organic molecule, notably glucose.

Effects of Local Conditions on Enzymes

Temp and pH: Can affect an enzyme’s activity in vivo; changes in temperature and pH can result in denaturing of the enzyme and loss of activity due to loss of 2°, 3°, or 4° structure.

Salinity: In vitro, salinity can impact the action of enzymes.

Regulation of Enzymes

Feedback Inhibition: An enzyme is inhibited by high levels of a product from later in the same pathway.

Reversible Inhibition: The ability to replace the inhibitor with a compound of greater affinity or to remove it using mild laboratory treatment.

Competitive Inhibition: When the inhibitor is similar to the substrate and binds at the active site, blocking the substrate from binding. Can be overcome by adding more substrate. Vmax is unchanged, Km increases.

Uncompetitive Inhibition: When the inhibitor binds only with the enzyme-substrate complex. Vmax and Km both decrease.

Noncompetitive Inhibition: When the inhibitor binds with equal affinity to the enzyme and the enzyme-substrate complex. Vmax decreases, Km is unchanged.

Mixed Inhibition: When the inhibitor binds with unequal affinity to the enzyme and the enzyme-complex. Vmax decreases, Km is increased or decreased depending on if the inhibitor has a higher affinity for the enzyme or enzyme-substrate complex.

Irreversible Inhibition: Alters the enzyme in such a way that the active site is unavailable for a prolonged duration or permanently.

Suicide Inhibitor: A substrate analogue that binds IRREVERSIBLY to the active site via a covalent bond.

Allosteric Effector: Binds at the allosteric site and induces a change in the conformation of the enzyme so the substrate can no longer bind to the active site. Displays cooperativity, so it does not obey Michaelis-Menten kinetics.

Homotropic Effector: An allosteric regulator that IS ALSO the substrate. Ex: O₂ is a homotropic allosteric regulator of hemoglobin.

Heterotropic Effector: An allosteric regulator molecule that is DIFFERENT from the substrate.

Phosphorylation: Covalent modification with phosphate.
Catabolism: Phosphorylated = active
Anabolism: Phosphorylated = inactive

Glycosylation: Covalent modification with carbohydrate.

Zymogens: Precursor to an enzyme. Secreted in an inactive form and are activated by cleavage.

Reaction scheme for Michaelis-Menten enzyme activity. To simplify things, we assume that almost none of the product reverts back to ES, which is true at the start of the reaction. This is why K-2 is omitted from the reaction scheme shown below.

Enzyme Kinetics

Saturation Kinetics: As [S] ↑ ⇒ ↑rxn rate, until a max value is reached.

Graphical Plots:
Michaelis-Menten: Hyperbolic curve
Lineweaver-Burk: Line

Km: The [S] at which an enzyme runs at half its Vmax
Km = (K-1 + K2) / K1

Vmax: The maximum rate at which an enzyme can catalyze a reaction. This is when all enzyme active sites are saturated with substrate.

Michaelis–Menten Equation:
V0 = (Vmax [S]) / ([S] + Km)

Cooperative Enzymes: Display a sigmoidal curve because of the change in activity with substrate binding.

Mechanisms of Enzyme Activity

Enzymes act by stabilizing the transition state, providing a favorable microenvironment, or bonding with the substrate molecules.

Active Site: The site of catalysis.

Lock & Key Theory: The enzyme and substrate are exactly complementary and fit together like a key into a lock.

Induced Fit Theory: The enzyme and substrate undergo conformational changes to interact fully.

Cofactors: Metal cation that is required by some enzymes.

Coenzyme: Organic molecule that is required by some enzymes.

Nonenzymatic Protein Function and Protein Analysis

Cellular Functions

Structural Proteins: Compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix. The most common structural proteins are collagen, elastin, keratin, actin, and tubulin. They are generally fibrous in nature.

Motor Proteins: Have one or more heads capable of force generation through a conformational change. They have catalytic activity, acting as ATPases to power mvmt. Common applications include muscle contraction, vesicle mvmt within cells, and cell motility. Examples include: myosin, kinesin, and dynein.

Binding Proteins: Bind a specific substrate, either to sequester it in the body or hold its concentration at steady state.

Cell Adhesion Molecules (CAM):
Cadherins: Calcium dependent glycoproteins that hold similar cells together.
Integrins: Have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix.
Selectins: Allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system.

Antibodies: Immunoglobulins, Ig. Used by the immune system to target a specific antigen, which may be a protein on the surface of a pathogen or a toxin. The variable region is responsible for antigen binding.

Protein Isolation

Electrophoresis: Uses a gel matrix to observe the migration of proteins in response to an electric field.

Native PAGE: Maintains the protein’s shape, but results are difficult to compare because the mass / charge ratio differs for each protein.

SDS-PAGE: Denatures the proteins and masks the native charge so that comparison of size is more accurate, but functional protein cannot be recaptured from the gel.

Isoelectric Focusing: Separates proteins by their isoelectric point (pI); the protein migrates toward an electrode until it reaches a region of the gel where pH = pI of the protein.

Chromatography: Separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase.

Column Chromatography: Uses beads of a polar compound (stationary phase) with a nonpolar solvent (mobile phase).

Ion-Exchange Chromatography: Uses a charged column and a variably saline eluent.

Size-Exclusion Chromatography: Relies on porous beads. Larger molecules elute first because they are not trapped in the small pores.

Affinity Chromatography: Uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest.

Biosignaling

Ion Channels: Can be used for regulating ion flow into or out of a cell.
Ungated Channels: Always open.
Voltage-Gated Channels: Open within a range of membrane potentials.
Ligand-Gated Channels: Open in the presence of a specific binding substance, usually a hormone or neurotransmitter.

Enzyme-Linked Receptors: Participate in cell signaling through extracellular ligand binding and initiation of 2nd messenger cascades.

G Protein-Coupled Receptors: GPCR has a membrane-bound protein called the G-Protein (α, β, γ subunits). The 1st messenger ligand initiates the 2nd messenger and the cascade response.

Protein Analysis

Structure: Primarily determined through x-ray crystallography after the protein is isolated, although NMR can also be used.

Amino Acid Sequence: Determined using the Edman Degradation.

Concentration: Determined colorimetrically, either by UV spectroscopy or through a color change reaction. Bradford Assay, BCA Assay, and Lowry Reagent Assay each test for protein and have different advantages and disadvantages. The Bradford Protein Assay is most common. It uses a color change from brown-green → blue.

Beer-Lambert Law:
Absorbance = ε C l
ε = extinction coefficient
C = concentration
l = path length in cm

Notes: Epinephrine is a ligand 1st messenger. At the end of the GPCR process, Phosphodiesterase deactivates cAMP and GTP hydrolyzed back to GDP.

Carbohydrate Structure and Function

Carbohydrate Classification

Nomenclature: 3 carbons: Trioses, 4 carbons: Tetroses, etc.
Some common names: glucose, fructose & galactose.

D and L: Based on the D- and L- forms of glyceraldehyde. Look at the highest numbered chiral carbon, -OH on right = D-sugars, -OH on left = L-sugars. Nearly all carbohydrates in nature are in the D-configuration. Compared to amino acids, which are found in the L-configuration.

Enantiomers: Stereoisomers that are non-superimposable mirror images of each other. D and L forms of the same sugar.

Diastereomers: Any stereoisomer that is not an enantiomer.
Epimers: Subtype of diastereomers that differ at exactly one chiral carbon.
Anomers: A subtype of epimers that differ at the anomeric carbon.

Monosaccharides

Monosaccharides: Single carbohydrate units, with glucose as the most commonly observed monomer. Can undergo oxidation/reduction, esterification, and glycoside formation.

Aldoses: Oxidized into aldonic acids, reduced to alditols.

Sugar as Reducing Agent: Sugars that can be oxidized are reducing agents themselves. Can be detected by reacting with Tollen’s or Benedict’s reagents.

Deoxy Sugars: -H replaces –OH

Esterification: Sugars react with carboxylic acids and their derivatives, forming esters.

Phosphorylation: A phosphate ester is formed by transferring a phosphate group from ATP onto the sugar. This rxn is similar to esterification.

Glycoside Formation: The basis for building complex carbohydrates and requires the anomeric carbon to link to another sugar.

Cyclic Sugar Molecules

Cyclization: Describes the ring formation of carbohydrates from their straight-chain forms.

Anomeric Carbon: The new chiral center formed in ring closure; it was the carbon containing the carbonyl in the straight-chain form.

α-anomers: Have the –OH on the anomeric carbon trans to the free –CH₂OH group.

β-anomers: Have the –OH on the anomeric carbon cis to the free –CH₂OH group.

Haworth Projections: Represent 3D structure of a monosaccharide.

Mutarotation: Spontaneous shift from one anomeric form to another with the straight-chain form as an intermediate.

Examples of Cyclic Sugar Molecules

Complex Carbohydrates

Disaccharides: Form as a result of glycosidic bonding between two monosaccharide subunits. Common examples: sucrose, lactose, maltose.

Polysaccharides: Formed by repeated monosaccharide or polysaccharide glycosidic bonding.

Cellulose: The main structural component for plant cell walls. Main source of fiber in human diet.

Starches: Main energy storage form for plants.
Amylose: Unbranched
Amylopectin: Branched

Maltose: (glucose + glucose with alpha glycosidic bond)

Lactose: (galactose + glucose with beta glycosidic bond)

Sucrose: (glucose + fructose with alpha glycosidic bond)

Lipid Structure and Function

Structural Lipids

Characteristics: Lipids are insoluble in water and soluble in nonpolar organic solvents.

Phospholipids: Amphipathic and form the bilayer of membranes. Contain a hydrophilic (polar) head and hydrophobic (nonpolar) tails. The head is attached by a phosphodiester linkage, and determines the function of the phospholipid.

Saturation: Saturation of the fatty acid tails determines the fluidity of the membrane. Saturated fatty acid = less fluid.

Glycerophospholipids: Phospholipids that contain a glycerol backbone.

Sphingolipids: Contain a sphingosine backbone. Many (but not all) sphingolipid are also phospholipids with a phosphodiester bond. These are sphingophospholipids.

Sphingomyelins: The major class of sphingophospholipids and contain a phosphatidylcholine or phosphatidylethanolamine head group. Part of the myelin sheath.

Glycosphingolipids: Attached to sugar moieties instead of a phosphate group. Cerebrosides have 1 sugar connected to sphingosine. Globosides have 2 or more.

Gangliosides: Contain oligosaccharides with at least 1 terminal N-acetylneuraminic acid (NANA).

Waxes: Contain long-chain fatty acids esterified to long-chain alcohols. Used as protection against evaporation and parasites in plants and animals.

Phospholipid: Polar head, nonpolar tails

Schematic of a phospholipid: May use glycerol or sphingosine for the backbone

Cerebroside: A type of glycolipid. Any lipid linked to a sugar is a glycolipid

Signaling Lipids

Terpenes: Odiferous steroid precursors made from isoprene. One terpene unit (monoterpene) contains 2 isoprene units.

Terpenoids: Derived from terpenes via oxygenation or backbone rearrangement. Odorous characteristics.

Steroids: Contain 3 cyclohexane rings and 1 cyclopentane.

Steroid Hormones: Have high-affinity receptors, work at low concentrations, and affect gene expression and metabolism.

Cholesterol: A steroid important to membrane fluidity and stability; and serves as a precursor to many other molecules.

Prostaglandins: Are autocrine and paracrine signaling molecules that regulate cAMP levels. Affect smooth muscle contraction, body temp, sleep-wake cycle, fever, pain.

Vitamins A, D, E, & K: Fat soluble vitamins
Vitamin A: Carotene, vision.
Vitamin D: Cholecalciferol, bone formation.
Vitamin E: Tocopherols, antioxidants.
Vitamin K: Phylloquinone & menaquinones. Forms prothrombin, a clotting factor.

Energy Storage

Triacylglycerols: Storage form of fatty acids. Contain 1 glycerol attached to 3 fatty acids by ester bonds. Very hydrophobic so do not carry additional water weight.

Adipocytes: Animal cells used specifically for storage of large triacylglycerol deposits.

Free Fatty Acids: Unesterified fatty acids that travel in the bloodstream. Salts of free fatty acids are soaps.

Saponification: The ester hydrolysis of triacylglycerols using a strong base like sodium or KOH.

Micelle: Can dissolve a lipid-soluble molecule in its fatty acid core, and washes away with water because of its shell of carboxylate head groups.

Triacylglycerol

DNA and Biotechnology

Nucleic Acid Structure

Nucleic acids are polymers of nucleotides. Types include Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA).

1° Structure: Linear sequence of nucleotides.

2° Structure: Interactions between bases within the same molecule. In DNA, the bases are held together by hydrogen bonds. 2° structure is responsible for the shape of nucleic acid.
RNA 2° structure has 4 basic elements: Loops, helices, bulges, and junctions. Loops include stem-loops (hairpin loops), tetraloops, and pseudoknots.

3° Structure: The location of the atoms in 3D space.

4° Structure: Interactions of nucleic acids with other molecules. Example: Chromatin interacting with histones.

Nucleic Acid Structure:

Includes DNA structure and RNA structure.ncludes DNA structure and RNA structure.

DNA Structure

DNA: Deoxyribonucleic Acid. A macromolecule that stores genetic information in all living organisms.

Nucleoside: 5-carbon sugar + nitrogenous base. NO PHOSPHATE groups.

Nucleotide: A nucleoside with 1 to 3 phosphate groups added. Nucleotides in DNA contain deoxyribose; in RNA they contain ribose. Adenine (A), Thymine (T), Guanine (G), Cytosine (C), Uracil (U). In RNA, U replaces T, so A pairs with U via 2 h-bonds.

Watson-Crick Model: Backbone of alternating sugar/phosphate groups. Always read 5’ → 3’. Two strands with antiparallel polarity wound into a double helix.

Nitrogenous Bases:
Purines: Adenine and Guanine. Made of two rings.
Pyrimidines: Cytosine, Thymine, Uracil. Made of one ring.

Chargaff’s Rules: # of Purines = # of Pyrimidines. A = T; C = G

B-DNA vs Z-DNA: Most DNA is B-DNA, forming a right-handed helix. Low concentrations of Z-DNA, with a zigzag shape, may be seen with high GC-content or high salt concentration.

Denatured / Denatured: Pulled apart
Reannealed: Brought back together

DNA Replication

DNA Replication: The process of producing an identical replica of a DNA molecule. Occurs in the S (synthesis) phase of the cell cycle.

DNA Repair

Oncogenes: Develop from mutations of proto-oncogenes, and promote cell cycling. May lead to cancer.
Oncogenes = stepping on gas pedal

Tumor Suppressor Genes: Code for proteins that reduce cell cycling or promote DNA repair.
Mutated Tumor Suppressor genes = cutting the brakes

Proofreading: DNA Polymerase proofreads its work and excises incorrectly matched bases. The daughter strand is identified by its lack of methylation and corrected accordingly.

Mismatch Repair: Occurs during G2 phase using the genes MSH2 and MLH1.

Nucleotide Excision Repair: Fixes helix-deforming lesions of DNA such as Thymine dimers. A cut-and-patch process. Excision Endonuclease.

Base Excision Repair: Fixes nondeforming lesions of the DNA helix such as cytosine deamination by removing the base, leaving apurinic/apyrimidinic (AP) site. AP Endonuclease then removes the damaged sequence, which can be filled in with the correct bases.

Eukaryotic Chromosome Organization

46 chromosomes in human cells. DNA is wound around histone proteins to form nucleosomes, which may be stabilized by another histone protein. As a whole, DNA and its associated histones make up chromatin in the nucleus.

Chromatin: Heterochromatin: Dark, dense, and silent
Euchromatin: Light, uncondensed, and expressed

Telomeres: Ends of chromosomes. Contain high GC-content to prevent unraveling of the DNA. During replication, telomeres are shortened, but this can be partially reversed by telomerase.

Centromeres: Located in the middle of chromosomes and hold sister chromatids together until they are separated during anaphase in mitosis. High GC-content to maintain a strong bond between chromatids.

Acrocentric Chromosome: When the centromere is located near one end of the chromosome and not in the middle.

Recombinant DNA and Biotechnology

Recombinant DNA: DNA composed of nucleotides from 2 different sources.

Hybridization: The joining of complementary base pair sequences.

Polymerase Chain Reaction (PCR):
Used to make many copies of a specific DNA region in vitro. The key ingredients of PCR are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.

RNA and the Genetic Code

The Genetic Code

Central Dogma: States that DNA is transcribed to RNA, which is then translated to protein.

Degenerate Code: Allows for multiple codons to encode for the same amino acid.

Start / Stop:
Initiation (start): AUG
Termination (stop): UAA, UAG, UGA

Wobble: 3rd base in the codon. Allows for mutations to occur without effects in the protein.
Wobble base pairings are less stable.

Point Mutations:

Frameshift Mutations: Result from a nucleotide addition or deletion, and change the reading frame of subsequent codons.

RNA: Similar to DNA except: Ribose sugar instead of deoxyribose. Uracil instead of thymine. Single stranded instead of double stranded.

3 Types of RNA:

Translation

tRNA: Translates the codon into the correct amino acid.

Ribosomes: Factories where translation (protein synthesis) occurs.

Initiation: Prokaryotes: When the 30S ribosome attaches to the Shine-Dalgarno sequence and scans for a start codon; it lays down N-formylmethionine in the P site of the ribosome.
Eukaryotes: When the 40S ribosome attaches to the 5’ cap and scans for a start codon; it lays down methionine in the P site of the ribosome.

Elongation: The addition of a new aminoacyl-tRNA into the A site of the ribosome and transfer of the growing polypeptide chain from the tRNA in the P site to the tRNA in the A site. The now uncharged tRNA passes into the E site before exiting the ribosome. The A site moves to the P site.

Termination: Occurs when the codon in the A site is a stop codon; release factor places a water molecule on the polypeptide chain and thus releases the protein.

Posttranslational Modifications: Folding by chaperones. Formation of quaternary structure. Cleavage of proteins or signal sequences.
Covalent addition of other biomolecules (phosphorylation, carboxylation, glycosylation, prenylation).

DNA Ligase: Fuse the DNA strands together to create one strand.

Transcription

Helicase: Unwinds the DNA double helix.

RNA Polymerase II: Binds to the TATA box within the promoter region of the gene (25 base pairs upstream from first transcribed base).

hnRNA: Collective term for the unprocessed mRNA in the nucleus.

Posttranscriptional Modification: The process in eukaryotic cells where primary transcript RNA is converted into mature RNA. Introns cut out.

Exons: Exit the nucleus and form mRNA.

Introns: Spliced out so they stay in the nucleus. Introns also enable alternative splicing.

Alternative splicing: Usually introns are cut away and exons remain, but alternative splicing might change that. A certain exon may be cut out, or an intron may stay. This allows the RNA segment to code for more than one gene.

5’ Cap and Poly-A tail: Added to the mRNA. The cap and tail stabilize mRNA for translation.

Prokaryotic Cells: Can increase the variability of gene products from one transcript though polycistronic genes. There are multiple translation sites within the gene which leads to different gene products.

Control of Gene Expression in Prokaryotes

Jacob-Monod Model: Explains how Operons work.

Operons: Inducible or repressible clusters of genes transcribed as a single mRNA.

Inducible Systems: Under normal conditions, they are bonded to a repressor. They are turned on when an inducer pulls the repressor off. Example: Lac operon.

Repressible Systems: Transcribed under normal conditions; they can be turned off by a corepressor coupling with the repressor and the binding of this complex to the operator site. Example: Trp operon.

Control of Gene Expression in Eukaryotes

Transcription: Search for promoter and enhancer regions in the DNA, then bind to the DNA and recruit RNA polymerase.

Promoters: Are within 25 base pairs of the transcription start site.

Enhancers: Are more than 25 base pairs away from the transcription start site.

Modification of chromatin structure affects the ability of transcriptional enzymes to access the DNA through histone acetylation (increases accessibility) or DNA methylation (decreases accessibility).

Biological Membranes

Fluid Mosaic Model

Accounts for the presence of lipids, protein, and carbohydrates in a dynamic, semisolid plasma membrane that surrounds cells.

Phospholipid: Each phospholipid has a hydrophilic head and hydrophobic tail. They are arranged so the heads are facing outward and the tails make up the inside of the membrane. Proteins are embedded in the bilayer.

Lipid Rafts: Lipids move freely in the plane of the membrane and can assemble into lipid rafts.

Flippases: Specific membrane proteins that maintain the bidirectional transport of lipids between the layers of the phospholipid bilayer in cells.

Proteins & Carbohydrates: May also move within the membrane, but are slowed by their relatively large size.

Membrane Components

Lipids: The primary membrane component, both by mass and mole fraction.

Triacylglycerols & Fatty Acids: Act as phospholipid precursors and are found in low levels in the membrane.

Glycerophospholipids: Replace one fatty acid with a phosphate group, which is often linked to other hydrophilic groups.

Cholesterol: Is present in large amounts and contributes to membrane fluidity and stability.
↑temp = INCREASE fluidity
↓temp = DECREASE fluidity

Waxes: Present in very small amounts, if at all; they are most prevalent in plants and function in waterproofing and defense.

Transmembrane Proteins: A type of integral protein that spans the entire membrane. They are often glycoproteins.

Embedded Proteins: Are most likely part of a catalytic complex or involved in cellular communication.

Membrane-Associated Proteins: May act as recognition molecules or enzymes.

Carbohydrates: Can form a protective glycoprotein coat and also function in cell recognition.

Ligands: Extracellular ligands can bind to membrane receptors, which function as channels or as enzymes in second messenger pathways.

Gap Junctions: Allow for rapid exchange of ions and other small molecules between adjacent cells.

Tight Junctions: Prevent solutes from leaking into the space between cells via a paracellular route, but do not provide intercellular transport.

Desmosomes & Hemidesmosomes: Desmosomes bind adjacent cells by anchoring to their cytoskeletons. Hemidesmosomes are similar, but their main function is to attach epithelial cells to underlying structures.

Membrane Transport

Concentration Gradients: All transmembrane movement is based on concentration gradients. The gradient tells us whether the process is passive or active.

Osmotic Pressure: A colligative property. It is the pressure applied to a pure solvent to prevent osmosis and is used to express the concentration of the solution. It can be conceptualized as a “sucking” pressure in which a solution is drawing water in, proportional to its concentration.
π = i M R T

Passive Transport: Does not require energy because the molecule is moving down its concentration gradient.

Simple Diffusion: A form of passive transport. Small, nonpolar molecules passively move from an area of high concentration to an area of low concentration until equilibrium is achieved.

Osmosis: A form of passive transport. Describes the diffusion of water across a selectively permeable membrane.

Facilitated Diffusion: A form of passive transport. Uses transport proteins to move impermeable solutes across the cell membrane.

Active Transport: Requires energy in the form of ATP or an existing favorable ion gradient.

Primary Active Transport: Uses ATP or another energy molecule to directly power the transport of molecules across a membrane.

Secondary Active Transport: “Coupled transport”. Harnesses energy released by one particle going down its electrochemical gradient to drive a different particle up its gradient.
Symport: Both particles flow the same direction.
Antiport: The particles flow in opposite directions.

Endocytosis & Exocytosis: Methods of engulfing material into the cells or releasing material out of the cell.
Pinocytosis: Ingestion of liquids via vesicles.
Phagocytosis: Ingestion of larger solid materials.

Specialized Membranes

Membrane Potential (Vm): Maintained by the Na+/K+ pump and leak channels. Resting potential of most cells is between −40 and −80 mV.

Nernst Equation: The electrical potential created by one ion can be calculated using the Nernst equation:
E = (RT / zF) ln ([ion]outside / [ion]inside) = (61.5/z) log ([ion]outside / [ion]inside)

Goldman-Hodgkin-Katz Voltage Eq: Resting potential of a membrane at physiological temp can be calculated using this equation, which is derived from the Nernst equation:
Vm = 61.5 log (PK[K+]outside + PNa[Na+]outside + PCl[Cl–]inside / PK[K+]inside + PNa[Na+]inside + PCl[Cl–]outside)

Mitochondrial Membrane:
The outer mitochondrial membrane is highly permeable to metabolic molecules and small proteins.
The inner mitochondrial membrane surrounds the mitochondrial matrix, where the citric acid cycle produces electrons used in the ETC. The inner mito membrane does not contain cholesterol.

Carbohydrate Metabolism I – Glycolysis, Glycogen, Gluconeogenesis, and the Pentose Pathway

Glucose Transport

GLUT-2: Found in liver (for glucose storage) and pancreatic β-islet cells (as part of the glucose sensor). Has high Km.

GLUT-4: Found in adipose tissue and muscle. Stimulated by insulin. Has low Km.

Glycolysis

Glucose + 2NAD+ + 2ADP + 2Pi → 2Pyruvate + 2ATP + 2NADH + 2H+

Important enzymes:

The NADH produced in glycolysis is oxidized by the mitochondrial electron transport chain when O2 is present. If O2 or mitochondria are absent, the NADH produced in glycolysis is oxidized by cytoplasmic lactate dehydrogenase. Examples include RBCs and skeletal muscle.

Pyruvate Dehydrogenase

A complex of enzymes that convert pyruvate to Acetyl-CoA right before the citric acid cycle. It is stimulated by insulin and inhibited by acetyl-CoA.

Glycogenesis and Glycogenolysis

Glycogenesis: The production of glycogen using two main enzymes: Glycogen Synthase, and Branching Enzyme. Occurs in the liver and muscle cells. Glycogen is stored in liver.

Glycogen Synthase: Creates α-1,4 glycosidic bonds between glucose.

Branching Enzyme: Creates branches with α-1,6 glycosidic bonds.

Glycogenolysis: The breakdown of glycogen using two main enzymes: Glycogen Phosphorylase, and Debranching Enzyme.

Glycogen Phosphorylase: Removes single glucose 1-phosphate molecules by breaking α-1,4 glycosidic bonds.
In the liver, it is activated by glucagon to prevent low blood sugar.
In exercising skeletal muscle, it is activated by epinephrine and AMP to provide glucose for the muscle itself.

Debranching Enzyme: Moves a block of oligosaccharide from the branch and connects it to the chain using an α-1,4 glycosidic bond. It also removes the branchpoint, which is connected via an α-1,6 glycosidic bond, releasing a free glucose molecule.

Gluconeogenesis

Occurs in both the cytoplasm and mitochondria, predominantly in the liver with a small contribution from the kidneys.
Most gluconeogenesis is simply the reverse of glycolysis, using the same enzymes.
The 3 irreversible steps of glycolysis must be bypassed by different enzymes.

Pyruvate Carboxylase: Converts pyruvate to oxaloacetate, which is converted to PEP by PEPCK.
Together, these two enzymes bypass pyruvate kinase.
Pyruvate carboxylase is activated by Acetyl-CoA. PEPCK is activated by glucagon and cortisol.

Fructose-1,6-bisphosphatase: Converts fructose 1,6-bisphosphate to fructose 6-phosphate, bypassing phosphofructokinase-1. This is the rate-limiting step of gluconeogenesis. It is activated by ATP and glucagon. Inhibited by AMP and insulin.

The Pentose Phosphate Pathway

Also known as the hexose monophosphate (HMP) shunt, it occurs in the cytoplasm of most cells.
Glucose 6-Phosphate enters the pathway and the products are NADPH, sugars for biosynthesis, and glycolysis intermediates.

Rate-Limiting Enzyme: Glucose-6-phosphate dehydrogenase (G6PD), which is activated by NADP+ and insulin and inhibited by NADPH.

Other Monosaccharides

Galactose: Comes from lactose in milk. Trapped in the cell by galactokinase, and converted to 1-phosphate via galactose-1-phosphate uridyltransferase and an epimerase.

Fructose: Comes from honey, fruit, and sucrose. Trapped in the cell by fructokinase, then cleaved by aldolase B to form glyceraldehyde and DHAP.

Carbohydrate Metabolism I – Aerobic Respiration

Acetyl-CoA

Acetyl-CoA: Contains a high-energy thioester bond that can be used to drive other reactions when hydrolysis occurs.

Acetyl-CoA Formation: Can be formed from fatty acids, which enter the mitochondria using carriers. The fatty acid couples with CoA in the cytosol to form fatty acyl-CoA, which moves to the intermembrane space. The acyl (fatty acid) group is transferred to carnitine to form acyl-carnitine, which crosses the inner membrane. The acyl group is transferred to a mitochondrial CoA to re-form fatty acyl-CoA, which can undergo β-oxidation to form acetyl-CoA.

Can also be formed from the carbon skeletons of ketogenic amino acids, ketone bodies, and alcohol.

Pyruvate Dehydrogenase (PDH): Oxidizes pyruvate, creating CO2; it requires thiamine pyrophosphate (vitamin B1, TPP) and Mg2+.

Dihydrolipoyl Transacetylase: Oxidizes the remaining two-carbon molecule using lipoic acid, and transfers the resulting acetyl group to CoA, forming acetyl-CoA.

Dihydrolipoyl Dehydrogenase: Uses FAD to reoxidize lipoic acid, forming FADH2. This FADH2 can later transfer electrons to NAD+, forming NADH that can feed into the electron transport chain.

Pyruvate Dehydrogenase Kinase: Phosphorylates PDH when ATP or acetyl-CoA levels are high, turning it off.

Pyruvate Dehydrogenase Phosphatase: Dephosphorylates PDH when ADP levels are high, turning it on.

Acyl Carnitine Translocase:

Mechanism for Acyl CoA to enter the mitochondrial matrix. The mitochondrial matrix is where Acyl CoA can undergo β-oxidation to form Acetyl-CoA.

Oxidative Phosphorylation

Proton-Motive Force:

The electrochemical gradient generated by the electron transport chain across the inner mitochondrial membrane. The intermembrane space has a higher concentration of protons than the matrix; this gradient stores energy, which can be used to form ATP via chemosmotic coupling.

ATP Synthase:

The enzyme responsible for generating ATP from ADP and Pi.

Reactions of the Citric Acid Cycle

Citric Acid Cycle: Takes place in the mitochondrial matrix. Its main purpose is to: oxidize carbons in intermediates to CO2 and generate high-energy electron carriers (NADH and FADH2) and GTP.

Citrate Synthase: Couples acetyl-CoA to oxaloacetate and then hydrolyzes the resulting product, forming citrate and CoA-SH. This enzyme is regulated by negative feedback from ATP, NADH, succinyl-CoA and citrate.

Aconitase: Isomerizes citrate to isocitrate.

Isocitrate Dehydrogenase: Oxidizes and decarboxylates isocitrate to form α-ketoglutarate. This enzyme generates the first CO2 and first NADH of the cycle. As the rate-limiting step of the citric acid cycle, it is heavily regulated: ATP and NADH are inhibitors; ADP and NAD+ are activators.

α-Ketoglutarate Dehydrogenase Complex: Acts similarly to PDH complex, metabolizing α-ketoglutarate to form succinyl-CoA. This enzyme generates the second CO2 and second NADH of the cycle. It is inhibited by ATP, NADH, and succinyl-CoA; it is activated by ADP and Ca2+.

Succinyl-CoA Synthetase: Hydrolyzes the thioester bond in succinyl-CoA to form succinate and CoA-SH. This enzyme generates the one GTP generated in the cycle.

Succinate Dehydrogenase: Oxidizes succinate to fumarate. This flavoprotein is anchored to the inner mitochondrial membrane because it requires FAD, which is reduced to form the FADH2 generated in the cycle.

Fumarase: Hydrolyzes the alkene bond of fumarate, forming malate.

Malate Dehydrogenase: Oxidizes malate to oxaloacetate. This enzyme generates the third and final NADH of the cycle.

The Electron Transport Chain

Electron Transport Chain: Takes place on the matrix-facing surface of the inner mitochondrial membrane. NADH donates electrons to the chain, which are passed from one complex to the next. As the ETC progresses, reduction potentials increase until oxygen, which has the highest reduction potential, receives the electrons.

Complex I: NADH-CoQ Oxidoreductase. Uses an iron-sulfur cluster to transfer electrons from NADH to flavin mononucleotide (FMN), and then to CoQ, forming CoQH2. 4 H+ ions are translocated by Complex I.

Complex II: Succinate-CoQ Oxidoreductase. Uses an iron-sulfur cluster to transfer electrons from succinate to FAD, and then to CoQ, forming CoQH2. No H+ pumping occurs at Complex II.

Complex III: CoQH2-Cytochrome c Oxidoreductase. Uses an iron-sulfur cluster to transfer electrons from CoQH2 to heme, forming cytochrome c as part of the Q cycle. 4 H+ ions are translocated by Complex III.

Complex IV: Cytochrome c Oxidase. Uses cytochromes and Cu2+ to transfer electrons in the form of hydride ions (H–) from cytochrome c to oxygen, forming water. 2 H+ ions are translocated by Complex IV.

NADH cannot cross the inner mitochondrial membrane. Therefore, one of two available shuttle mechanisms to transfer electrons in the mitochondrial matrix must be used.

Glycerol 3-Phosphate Shuttle: Electrons are transferred from NADH to DHAP, forming glycerol 3-phosphate. These electrons can then be transferred to mitochondrial FAD, forming FADH2.

Malate-Aspartate Shuttle: Electrons are transferred from NADH to oxaloacetate, forming malate. Malate can then cross the inner mitochondrial membrane and transfer the electrons to mitochondrial NAD+, forming NADH.

Lipid and Amino Acid Metabolism

Lipid Digestion and Absorption

Mechanical Digestion: Mechanical digestion of lipids occurs primarily in the mouth and stomach.

Chemical Digestion: Chemical digestion of lipids occurs in the small intestine and is facilitated by bile, pancreatic lipase, colipase, and cholesterol esterase.

Emulsification:Upon entry into the duodenum, emulsification occurs, which is the mixing of two normally immiscible liquids; in this case, fat and water. (A common example of an emulsion is oil-and-vinegar salad dressing). This increases the surface area of the lipid, which permits greater enzymatic interaction and processing. Emulsification is aided by bile salts.

Micelles: Water-soluble spheres with a lipid-soluble interior. Digested lipids may form micelles to be carried to the intestinal epithelium where they are absorbed across the plasma membrane.

Short vs. Long Chain Fatty Acids: Short-chain fatty acids are absorbed across the intestine into the blood. Long-chain fatty acids are absorbed as micelles and assembled into chylomicrons for release into the lymphatic system.

Lipid Mobilization and Transport

Lipid Mobilization: Lipids are mobilized from adipocytes by hormone-sensitive lipase. Lipids are mobilized from lipoproteins by lipoprotein lipase.

Chylomicrons: Transport dietary triacylglycerols, cholesterol, & cholesteryl esters from intestine to tissues. Uses the lymphatic system.

Lipoproteins:

The transport mechanism for lipids.
Very-low-density: Liver → tissues.
Intermediate-density: Transition particle. VLDL → IDL → VLDL
Low-density: Bad. Moves cholesterol → tissues.
High-density: Good. Moves cholesterol → liver, exits body.

Apolipoproteins: Form the protein component of lipoproteins. They are receptor molecules that control interactions between lipoproteins.

Cholesterol Metabolism

Cholesterol may be obtained through dietary sources or through de novo synthesis in the liver

HMG-CoA Reductase: Synthesizes mevalonate. This is the rate limiting step of cholesterol synthesis.

LCAT: Catalyzes the formation of cholesteryl esters for transport with HDL

CETP: Catalyzes the transition of IDL to LDL by transferring cholesteryl esters from HDL

Fatty Acids and Triacylglycerols

Fatty Acids:

Carboxylic acids with a long chain
Saturated: No double bonds
Unsaturated: One or more double bonds

Fatty Acid Synthesis: Synthesized in cytoplasm from acetyl-CoA transported out of the mitochondria. Five steps: Activation, bond formation, reduction, dehydration, and a second reduction.

Arachidonate: Precursor to eicosanoid signaling molecules: prostaglandins, prostacyclins, & thromboxanes. Also precursor to leukotrienes.

Fatty Acid Oxidation:Oxidation occurs in the mitochondria following transport by the carnitine shuttle. β-oxidation uses cycles of oxidation, hydration, oxidation, and thiolysis cleavage. The fatty acid chain is shortened by two carbon atoms. FADH2, NADH, and acetyl CoA are generated.

The carboxylic acid is the α end. The ω carbon counting starts on the other end.

Cis-oleate, an cis-Δ9 fatty acid
The cis bond prevents tight packing, which lowers the melting point.

α-Linolenate, an Omega-3 Fatty Acid
(3rd carbon from the ω end)

Ketone Bodies

Ketogenesis: Ketone bodies form via ketogenesis due to excess acetyl-CoA in the liver during a prolonged starvation state

Ketolysis: Regenerates acetyl-CoA for use as an energy source in peripheral tissues

Energy Source: The brain can derive up to 2/3 of its energy from ketone bodies during prolonged starvation

Protein Catabolism

Protein digestion occurs primarily in the small intestine. Catabolism of cellular proteins occurs only under conditions of starvation. Amino acids released from proteins usually lose their amino group through deamination. The remaining carbon skeleton can be used for energy.

Glucogenic Amino Acids: Can be converted into glucose through gluconeogenesis. All but leucine and lysine.

Ketogenic Amino Acids: Can be converted into acetyl-CoA and ketone bodies. Leucine and lysine are the only amino acids that are solely ketogenic.

Fates of the amino acid carbon skeleton following protein catabolism

Bioenergetics and Regulation of Metabolism

Thermodynamics and Bioenergetics

Open System: Matter & energy can be exchanged with the environment

Closed System: Only energy can be exchanged with the environment. No work is performed because pressure and volume remain constant. Enthalpy = Δinternal energy = heat exchange

Entropy: A measure of energy dispersion in a system

Change in Free Energy:

Standard Free Energy, ΔG°: The energy change that occurs at 1 M concentration, 1 atm, and 25°C.
Modified Standard State, ΔG°’: Indicates physiological conditions. [H+] = 10-7 M, so pH is 7.

The Role of ATP

ATP is a mid-level energy molecule. It contains high-energy phosphate bonds that are stabilized upon hydrolysis by resonance, ionization, and loss of charge repulsion.

Energy Source: ATP provides energy through hydrolysis and coupling to energetically unfavorable reactions. ATP = 30 kJ/mol

Phosphoryl Group Transfers: ATP can donate a phosphate group to other molecules.

Group Transfers: For example, in Glycolysis, it donates a Phosphate group to glucose to form glucose 6-phosphate

Biological Oxidation and Reduction

Biological oxidation and reduction reactions can be broken down into component half-reactions. Half-reactions provide useful information about stoichiometry and thermodynamics

High Energy Electron Carriers: May be soluble or membrane-bound. Includes NADH, NADPH, FADH2, ubiquinone, cytochromes, and glutathione.

Flavoproteins: A subclass of electron carriers that are derived from riboflavin (vitamin B2). Examples: FAD and FMN

Metabolic States

Equilibrium: Equilibrium is an undesirable state for most biochemical reactions because organisms need to harness free energy to survive.

Postprandial State: Well-fed, absorptive. ↑ Insulin. Anabolism prevails.

Postabsorptive State: Fasting. ↓ Insulin. ↑ Glucagon and catecholamine. Transition to catabolism.

Prolonged Fasting: Starvation. ↑ Glucagon and catecholamine. Most tissues rely on fatty acids. 2/3 of brain activity can be derived from ketone bodies.

Integrative Metabolism

Calorimetry: Measures metabolic rates

Respiratory Quotient: Estimates the composition of fuel that is actively consumed by the body. RQ = CO2 produced / O2 consumed

Regulatory Hormones:

Ghrelin: ↑ appetite. (sight, sound, taste, smell of food)
Orexin: ↑ appetite.
Leptin: ↓ appetite by suppressing orexin production

Body Mass Index:

BMI = mass / height2

Hormonal Regulation of Metabolism

Insulin:

Secreted by pancreatic β-cells, regulated by glucose
↓ blood glucose by increasing cellular uptake
↑ rate of anabolic metabolism

Glucagon:

Secreted by pancreatic α-cells, stimulated by low glucose and high amino acid levels
↑ blood glucose by promoting gluconeogenesis and glycogenolysis in the liver

Glucocorticoids:

Blood glucose in response to stress by mobilizing fat stores and inhibiting glucose uptake. They increase the impact of glucagon and catecholamines. Ex: cortisol

Catecholamines:

Promote glycogenolysis and ↑ basal metabolic rate through sympathetic nervous system activity.
“Adrenaline rush.” Ex: epinephrine and norepinephrine

Thyroid Hormones:

↑ basal metabolic rate, as evidenced by O2 consumption and heat production when they are secreted. T3 is more potent than T4, but has a shorter half-life and is available in lower concentrations in the blood. T4 is converted to T3 at the tissues. Thyroid hormones are tyrosine-based.

Tissue-Specific Metabolism

Liver: The most metabolically diverse tissue. Hepatocytes are responsible for the maintenance of blood glucose levels by glycogenolysis and gluconeogenesis in response to pancreatic hormone stimulation. The liver also processes lipids and cholesterol, bile, urea, and toxins.

Adipose Tissue: Stores lipids under the influence of insulin and releases them under the influence of epinephrine.

Skeletal Muscle: Skeletal muscle metabolism will differ depending on current activity level and fiber type.

Cardiac Muscle: Uses fatty acid oxidation in both the well-fed and fasting states.

Brain and Nervous Tissue: Consume only glucose in all metabolic states, except for prolonged fasts, where up to 2/3 of the brain’s fuel may come from ketone bodies.