Biochemistry: Amino Acids and Non-Covalent Bonds

• Amino acids are composed of an alpha-carbon, a carboxyl group (-COOH), an amino group (-NH2), a hydrogen atom, and a functional side chain (R-group).
• All amino acids, except glycine, are chiral; human proteins contain L-amino acids.
• Amino acids can be classified as non-polar aliphatic, aromatic, polar uncharged, positively charged, or negatively charged.
• Non-polar aliphatic amino acids include Gly, Ala, Val, Leu, Ile.
• Aromatic amino acids include Phe, Tyr, Trp.
• Polar uncharged amino acids include Ser, Thr, Cys, Asn, Gln.
• Positively charged amino acids include Lys, Arg, His.
• Negatively charged amino acids include Asp, Glu.
• The hydrophobic effect is the key force driving protein folding and interaction.
• Amino acids act as buffers, and their pKa influences their protonation states.
• Essential amino acids must be obtained through diet.

Non Covalent Interactions

• Hydrogen bonds involve a hydrogen donor (O-H, N-H) and a hydrogen acceptor (O, N).
• Linear hydrogen bonds are strongest and have an angle of 180°.
• Ionic (Coulombic) interactions involve attraction/repulsion between charged groups and are electrostatic.
• Van der Waals forces are weak dipole-dipole interactions dependent on distance.
• Hydrophobic effect involves the ordering of H2O around non-polar molecules.

Peptide Bonds

• Peptide bond formation is a condensation reaction requiring energy.
• Peptide bonds exhibit slow hydrolysis; degradation requires proteases.
• Isopeptide bonds form between side chains, such as in Lys-Glu ubiquitination.

Post-Translational Modifications

• Phosphorylation occurs at Ser, Thr, Tyr and is involved in signal transduction and enzyme regulation.
• Glycosylation occurs at Asn, Ser, Thr and affects protein stability, sorting, and interactions.
• Acetylation occurs at Lys (N') and regulates gene transcription.
• Methylation occurs at Lys, Arg and is involved in epigenetic regulation.
• Disulfide bridges form between Cys residues, stabilizing extracellular proteins.
• Lipidation occurs at Cys-Gly and is involved in membrane localization.

Selenocysteine

• Selenocysteine (Sec, U) is the 21st amino acid, incorporated with UGA, which is important in redox reactions.
• Hydroxyproline and Hydroxylysine are found in collagen and are essential for its stability.
• Gamma-Carboxyglutamate is key in blood clotting factors.

Protein Structure and Function

• Primary structure is the amino acid sequence.
• Secondary structure is stabilized by hydrogen bonds.
• Tertiary structure is influenced by hydrophobic effect and disulfide bonds.
• Quaternary structure involves multiple subunits.

Biomolecular Interactions

• Stabilizing bonds and interactions in biomolecules.
• Covalent bonds are strong and formed by sharing electron pairs.
• Peptide bonds link amino acids in proteins.
• Disulfide bonds between two Cys residues stabilize proteins.

Non-Covalent Bonds

• Non-covalent bonds are weaker and reversible.
• Hydrogen bonds involve a hydrogen donor and acceptor and are essential for secondary structure and DNA base pairing.
• Ionic interactions occur between oppositely charged groups.
• Van der Waals forces are weak attractions due to temporary dipoles.
• Hydrophobic interactions involve nonpolar amino acid side chains clustering together in aqueous environments for protein folding and membrane formation.

Amino Acids: Structure, Properties

• Amino acids have an α-carbon, NH2, H, COOH, and an R group.
• R groups can be nonpolar hydrophobic, polar uncharged, positive charged (basic), or negative charged (acidic).
• Cysteine contains a thiol (-SH) group and forms disulfide bonds.
• Histidine has an imidazole group and acts as a proton donor/acceptor at physiological pH.
• Tyrosine, Tryptophan, and Phenylalanine are aromatic and absorb UV light.

Selenocysteine Uniqueness

• Selenocysteine contains selenium (Se) instead of sulfur (S), like cysteine.
• Selenium makes Selenocysteine more reactive in redox reactions.
• The encoding of Selenocysteine is incorporated via UGA and SECIS in mRNA and requires a unique tRNA.
• Selenocysteine is found in selenoproteins, which include enzymes like glutathione peroxidase (antioxidant defense) and iodothyronine deiodinase (thyroid hormone metabolism).

Peptide Bonds

• Peptide bond formation occurs via a condensation reaction, where the COOH of one amino acid reacts with the -NH2 of another, releasing H2O.
• This reaction is catalyzed by ribosomes during protein synthesis.
• Peptide bonds are planar and rigid due to partial double-bond character, limiting rotation.

Isopeptide Bonds

• Isopeptide bonds are a type of amide bond that forms between side chains instead of main groups.
• Common examples include ubiquitination (lysine with C' of carboxyl of ubiquitin) and blood clotting (Factor XIIIa catalyzes a bond between Lys and Gln residues in fibrin cross-linking).
• These bonds are important in protein regulation, stability, and PTMs.

Protein Structure

• Primary structural hierarchy involves a linear arrangement.
• Secondary structural elements are localized folding patterns that are not for all proteins.
• Alpha-helices (intrachain H bonds) and beta-sheets (H bonds by adjacent strands) exist.
• Beta-turns, with direction changes, are common to Proline and Glycine.
• Tertiary structure involves an overall 3D shape, stabilized by hydrophobic interactions, ionic bonds, H bonds, and disulfide bridges.
• Quaternary structure involves the assembly of multiple polypeptide subunits.

Peptide Bond: Structure

• Formation of a peptide bond occurs via condensation between NH2 and COOH, releasing H2O.
• Planar and restricts rotation.
• Peptide bonds have partial double bond character, which create a dipole.
• Peptide bonds have a trans configuration, except for Proline (can be cis).

Protein Folding/Denaturation

• Protein folding is a process to achieve the lowest free energy state and is not random.
• Hydrophobic effects are important for protein folding
• Chaperones interact with newly or improperly folded polypeptides (Hsp70, Hsp90).
• Chaperones prevent aggregation or degradation.
• Hsp70 uses ATP hydrolysis to stabilize proteins, and has multiple cycles of binding and release.
• Hsp60 has two chambers that provide a microenvironment, where closing the cap can lead to conformational changes.
• Folding and unfolding sometimes requires peptidyl-prolyl isomerase, which converts trans Pro to cis Pro.

Protein Denaturation

• Denaturation is caused by heat, pH, chaotropic agents (urea etc.), and organic solvents.
• Denaturation affects every organic molecule until the peptide bonds are disrupted.

Fibrous Proteins

• Fibrous proteins provide structural support.
• Collagen is composed of a triple helix stabilized by interchain H bonds.
• Collagen is also rich in glycine, proline, and hydroxyproline.
• Hydroxylation of proline depends on Vitamin C, and cross-linking strengthens the fibers.
• Keratin is composed of coiled-coil α-helices stabilized by disulfide bonds.

Globular Proteins

• Globular proteins are compact, spherical shapes that have diverse roles.
• Myoglobin stores O2 in muscle cells.
• It contains a heme group for O2 binding (prosthetic group) and is mainly alpha-helices.
• Myoglobin binds O2 too tightly to be useful in O2 transport.
• Hemoglobin is an O2 transport protein in RBCs.
• the binding of O2, increases the affinity for subsequent O2 molecules.
• Hemoglobin exists in T (tense) state, which exhibits low affinity.
• Hemoglobin also exists in R (Relaxed) state, which is high affinity.
• Carbamate forms additional salt bridges that stabilize the T state.

Enzymes

• Enzymes are biocatalyzers that increase reaction rates without being used up.
• Enzymes provide advantages over non-bio catalysts; enzymes specify stereospecifity, occur under mild conditions and allow regulation.

Enzyme Classes

• Oxidoreductases transfer electrons (e-), with cofactors such as NAD+/NADP+.
• Transferases transfer a functional group, and use transaminases, kinases + cofactors.
• Hydrolases cleave bonds by adding H2O, using proteases, lipases, nucleases.
• Lyases cleave bonds by other means, using aldolases and decarboxylases.
• Isomerases rearrange existing atoms, using mutases, epimerases + cofactors.
• Ligases catalyze condensation reactions coupled to ATP hydrolysis (+ cofactors).

Catalytic Activity

• Enzymes accelerate the rate by lowering the activation energy (ΔG‡).
• Enzymes do not alter the actual ΔG.
• Factors affecting enzymes are temperature and pH.

Catalysis Mechanisms

• Proximity and orientation reduce the entropy cost.
• Induced fit: conformational changes lead to optimization of the active site.
• Acid-base catalysis: specific amino acid residues in the active site donate/accept protons, facilitating bond breaking/formation.
• Covalent catalysis: a transient covalent bond may form between the enzyme and the substrate.
• Metal ion catalysis: Mg+2, Zn+2 etc. can stabilize negative charges or participate directly in the catalytic mechanism.
• Strain and transition state stabilization: enzymes impose strain on substrate bonds or stabilize the high energy transition state through complementary interactions.

Enzyme Kinetics/Inhibition.

• Michaelis-Menten Kinetics.
• Km (Michaelis Constant): substrate concentration at which the reaction rate is half of Vmax, reflects the affinity of the enzyme for its substrate.
• Vmax: the maximum rate achieved by the system at saturating substrate concentration.
• Kcat (Turnover number): number of substrate molecules converted to product per enzyme molecule per unit time when fully saturated.
• Catalytic efficiency equals kcat / Km (reflects affinity and rate).

Inhibition Mechanisms

• Competitive inhibition: inhibitor resembles the substrate, increases Km, no effect on Vmax.
• Noncompetitive inhibition: inhibitor binds to allosteric site, decreases Vmax, no effect on Km.
• Uncompetitive inhibition: inhibitor only binds to E-S complex, decreases both Km and Vmax.
• Suicide inhibition: inhibitor forms a covalent bound complex with the enzyme (E), permanently deactivating it.

Enzyme Regulation

• Allosteric regulation: enzymes with multiple subunits may exhibit cooperative binding.
• Allosteric regulation has S-shaped kinetics rather than hyperbolic M-M.
• Binding events at an "allosteric site" in a multiple subunit enzyme may lead to allosteric inhibition or activation, which is important in controlling metabolic pathways.
• Feedback inhibition: end products inhibit earlier enzymes/steps.
• Proteolytic activation: many enzymes are synthesized as inactive precursors (zymogens) and require specific cleavage to become active, such as digestive enzymes.
• Isozymes: different forms of an enzyme that catalyze the same reaction but differ in kinetic properties, regulation, tissue distribution, or response to inhibitors.

Cofactors

• Cofactors are non-protein chemical compounds.
• Cofactors can be inorganic ions like Mg and Zn or organic molecules known as coenzymes like NAD, FAD.

Catalytic Systems

• Ribozymes are RNA molecules with catalytic activity.
• Catalytic antibodies are engineered antibodies that act as enzymes.

Catalytic Triad

• Trypsin is a proteolytic enzyme secreted by the pancreas that has a highly reactive Serine residue.
• The catalytic triad consistes of Serine, Histidine, and Aspartate.

Cardiac Metabolism

• 90% ATP demand is powered by Oxphos.
• Prefers fatty acids (60-80%), also use glucose (10-40%), lactate.

Metabolic Pathways

• Glycolysis converts glucose to pyruvate that generates ATP and NADH .
• Gluconeogenesis generates glucose when glucose levels are low, some intermediates are used to generate glucose.
• Glycogen metabolism stores glucose as glycogen.
• Glycogen is broken down to release glucose 6-phosphate when needed.
• Oxidative pathway converts pyruvate into acetyl Co-A, feeding into the citric acid cycle.
• NADH and FADH2 facilitate ATP synthesis via the electron transport chain.
• Glucose is phosphorylated by hexokinase to ensure it stays inside the cell by adding a negative charge (P-), lowering activation energy and destabilizing the molecule.
• GLUT transporters can transport in both directions.

Glucose Uptake

• Carrier-mediated active transport.
• In the Apical membrane is a active transport because SGLT 1 uses the Na+ gradient to uptake glucose against its concentration gradient.
• In the Basolateral membrane, GLUT 2 mediates the movement of glucose from the gut to the bloodstream.
• GLUT 2 carriers both ways.
• Diffusion is facilitated.

Glucose Transporters

• GLUT 1/3 provides glucose uptake in most tissues.
• GLUT 2 is found in the liver, gut, pancreatic cells, and kidney.
• Higher Km to sense blood glucose levels better.
• GLUT 4 is located in Sk. muscle and adipose tissue and translocated to the plasma membrane in response to insulin.
• Transporters’ Km is low, this is good to maintain glucose levels inside the cells, even if glucose levels are low in the bloodstream.
• Km for GLUT2 is very high, this is good to store glycogen in the liver and take up glucose only when the blood levels are very high.

Hormonal Regulation

• Insulin is secreted by pancreatic β-cells and promotes glucose uptake, glycogen synthesis, and overall anabolic processes.
• Insulin is synthesized as preproinsulin, converted to proinsulin, and cleaved to mature insulin (with C-peptide).
• Glucagon is secreted by pancreatic α-cells that raises blood glucose by stimulating glycogenolysis and glucogeralysis, particularly in the liver.

Insulin Secretion

• Glucose enters the pancreatic β-cell via GLUT2.
• Glucose has glycolysis and ATP is obtained.
• High ATP closes K+ channels, causing the extrocellular membrane to depolarize.
• Due to depolarization, Ca+2 outside the cell goes inside, which elicits more Ca+2 secretion.
• Excess Ca+2 aids in insulin secretion, outside of the β-cell.

Glucose Uptake

• Increased glucose uptake through insulin-dependent transport of GLUT4 to the plasma membrane in myocytes and fat cells.
• The insulin step is rate-limiting.
• Insulin binds to the insulin receptor.
• Signal transduction delivers GLUT4 to the cellular membrane.
• Occurs to ensure glucose is taken up by these tissue types.

Glycogen Metabolism

• Synthesis forms an α-1,4 glycosidic bond with glycogen synthase, using UDP-glucose as the donor to an already existing glycogen molecule.
• Degradation cleaves α-1,4 bonds with glycogen phosphorylase via phosphorylation, producing glucose 1-phosphate.
• Branching enzyme creates highly branched soluble structures with α-1,6 bonds.
• Branching allows an increase in rapid synthesis and degradation.
• Debranching removes branches that glycogen phosphorylase can not process.
• Debranching is activated by phosphorylase kinase, which is activated by phosphorylation and Ca+2.
• Regulation of glycogen metabolism involves many ways to do this, for example, Glucagon (PKA pathway) and Insulin (PKB pathway).

Glycolysis

• Glycolysis comes in two phases, which are preparator and energy payoff.
• Glucose is phosphorylated to glucose-6-phosphate via hexokinase. ATP is required.
• Isomerization: G6P is rearranged into fructose-6-phosphate.
• Phosphorylation creates an irreversible reaction.
• F6P is phosphorylated to form fructose-1,6-bisphosphate with 1 ATP (Key Regulatory Step - PFK1)
• 1,6BPFK-1 is regulated by allosteric effectors like ATP, AMP, fructose 2,6-bp.
• Cleavages make 71,6 BP: the 6C molecule split into two 3C sugars, DHAP and GAP.
• DHAP is converting into GBP So that both can enter the later steps.

Fructose Chemistry

• The fructose group is required for its Schiff base in the Keto group.
• It helps to weaken the bond between C3 and C4.
• G3P Dehydrogenase: G3P from GAP is oxidized and phosphorylated to 1,3-bisphosphoglycerate while creating NAD to NADH.
• Substrate level phosphorylation is when 1,3BPG donates a phosphate group to ADP to form ATP, producing 3-phosphoglycerate.
• Phosphoendpyruvate transfers its phosphate to AIDP in a reaction catalyzed by pyruvate kinase, yielding a second molecule of ATP per G3P.
• It produces a reversible reaction.
• First substrate phosphorylation produces ATP.
• It depends on an enzymatic reaction.

Glycolysis (Steps)

• There are a number of key regulatory or irreversible steps.
• The steps serve as control points.
• To trap the glucose inside the cell phosphorylation by hexokinase is used.
• In most tissues allosteric product inhibition is used.
• In liver an B-cells regulation by glucose concentration is used. Phosphofructokinase acts as a rate-limiting step that sensitive to energy states.
• AMP Is used to describe ATP
• Fructose describes BDP
• Citrate describes H+
• Tissue Specific regulation are described by;
• In a liver the regulation is hormone driven, that helps to regulate between a switch
• Muscle is described by energy demand. It can camp up fast. →In cardiomyocytes: The kinase is never shut off by Gucagon since they always need energy.
• Pyruvate Kinase catalyzes the final step, ensuring the flow to create APT and pyruvate.
• Pyruvate kinase produces; atp, Alanin, Fructose is used to 1,6-bisphosphate
• NAD+ is the needed to be recovered.
• Glycolysis consumes 2 NAD.
• Glucose + 2P1 + 2 ADP + 2 NAD -> 2 Pyrwate + 2 ATP + 2 NADH + 2H+ + 2H2O.

NADH Conditions.

• In aeorbic conditions Oxidation of occurs.
• 2 NADH + 2H+ + 202 → 2NAD+ + 2 H20
• Pyruvate is found in erythocytes, and the heart and liver.

Lactose Cycle

• Lactose is the interogan exchange of the body for lactate.
• Lactose has myocytes and 02
• Myocites has glucoset, which supplies muscle glucese. → pyruvate dehydrogenase. It can be found in the matrix of mitochondria
• Pyruvate goes to accetyl for matrix.
• Accetyl can also be used, which is similar to ACC by Oxidation.

Electron Transport Chain:

• Citrate Synthesis goes two oxidation steps. NADH and FAXH2 can be created after.
• Third substrate level phosphorylation goes to. Into Succinate with succinyl .CoA AMP, and NADH for conversion of Pyruvate.. ++ СA (muscle) → insulin is a protein that can be used in the liver. Oxphos takes place on the inner membrane.
• complex I and II are where NAD and FAD enter; those products enter.
• Cytocrhome can then enter the complex, which is where complex = final enters.
• They create all complexes which make the PH acidic by the creation Electrochemical gradient.
• ATP synthesis
• Only 13 protiens sub units need them.
• The others make all the subunits. prosetehic- fad partners in redox reaction Q - freeely movable Cu is involved

F1-F0 ATP Synthase

• DNP is an uncouple and the cell becomes acidified
• DNP uncouoples to make heat
• DNP depletes.
• Thermoogenin brown creates cells
• BROWN is for u couple is how cells go forward

Liver

• Protein Amino acids
  Glucogenesis glucose Alanine alanive+ 02 alanine Animo Transerase

Amino Acid catbolism

• Aas are not storted but can turn into liver.
• Removing there a group Alpha groups help with energy group
• Transmaninatron -transfer aa from a acid and produce acid but a new amino. → use pLp with the cofactor forms base linking the enzyme and
• Aacids are created with lysine to create phosphate and phosphate for amino.
• Overal is to create aacids to link 2 group . aa acids go without use Aa goes direct in

Aa that come go and can

Fate of skeleton once create and cycle and produce aa or nucleo. Prouduce biogenc Lysine and dop aa are 4 group liver Most alanine Is alanine and glutmaine Removal of

• Remove is n pH needs to be home
• Liver help preent a bas → use bicarbamate - Kid prevent can a acid → Use amohya

→Anacletinc is made citrate 7 for point Aa

• For kid to make to the ketos AA Help make gluco