Protein Metabolism Notes

NOTES NOTES PROTEIN METABOLISM AMINO ACIDS & PROTEIN FOLDING osms.it/amino-acids-protein-folding Figure 4.1 The 20 amino acids used by humans. ▪ Amino acids: organic compounds with -NH2, -COOH groups ▪ Side chain gives specific properties ▫ Hydrophilic: polar side chains → acidic (e.g. carboxyl); basic (e.g. amine) ▫ Hydrophobic: non-polar side chains → alkyl, aromatic ▪ Molecular charge depends on pH ▫ Low pH: + amine, 0 carboxyl ▫ High pH: - carboxyl, 0 amine ▫ Neutral: + amine, - carboxyl → zwitterion ▪ Zwitterion: compound with both positive, negative charges ▫ Occurs in each amino acid at specific pH (AKA pI/isoelectric point) Figure 4.2 Amino acid structure. OSMOSIS.ORG 29

▪ Proteins: amino acid chains connected by peptide bonds ▫ Peptide bond: amide bond formed between amino acids by condensation of -NH2 with -COOH → releases H2O ▫ Resonance: electrons shared across bond → partial double-bond character → improved strength ▪ Amino acids: chiral molecules ▫ Enantiomers/mirror images are distinct ▫ Proteins only made of L-amino acids ▪ Protein production occurs in ribosomes Primary, secondary, tertiary, quaternary protein structures ▪ Primary: linear amino acid sequence connected by peptide bonds ▪ Secondary: α-helix, β-pleated sheet ▪ Tertiary: overall shape, including secondary structures, with other features (e.g. disulfide bridge, hydrophobic bonds) ▪ Quaternary: final level; combination of multiple amino acid chains (e.g. hemoglobin) Figure 4.3 Amide (peptide) bonds form between amino acids through a condensation reaction. Figure 4.4 Enantiomers are two forms that look like mirror images but are not interchangeable, like a left and right shoe. Proteins are only made out of levo-oriented amino acids. 30 OSMOSIS.ORG Figure 4.5 The four levels of structure for proteins.

Chapter 4 Biochemistry: Protein Metabolism ENZYME FUNCTION osms.it/enzyme-function ▪ Enzymes: biochemical reaction catalysts ▪ Substrates bind to active site → enzymesubstrate complex ▪ Not used up in reactions ▪ Highly specific (e.g. amylase in saliva → large carbohydrate breakdown) Figure 4.7 Michaelis–Menten graph: used to visualize enzyme kinetics. With a fixed amount of enzyme, the reaction velocity ↑ as substrate is added, until the active sites on all of the enzymes become saturated. At this point, the reaction speed plateaus → Vmax. ▪ Km: substrate concentration when reaction velocity is half of maximum ▫ ↑ enzyme affinity (e.g. activator molecules) → ↓ Km ▫ ↓ enzyme affinity (e.g. competitive inhibition) → ↑ Km Figure 4.6 Transition state: intermediate step in reaction with high energy. Enzymes speed up reactions by binding substrate (enzymesubstrate complex), which stabilizes the transition state and decreases the amount of extra energy required for the reaction to proceed. ▪ Enzyme kinetics: catalysis rate ▪ Vmax: maximum reaction velocity with fixed enzyme quantity ▫ ↑ substrate → ↑ velocity, until all enzymes bind ▫ ↑ enzymes → ↑ Vmax ▫ ↓ enzymes → ↓ Vmax ▫ Non-competitive inhibition (inhibitory molecule binds to active/allosteric site → prevents substrate binding) → ↓ Vmax Figure 4.8 Km is found using a Michaelis– Menten diagram by identifying ½ Vmax on the y-axis, then finding the corresponding substrate concentration value on the x-axis. OSMOSIS.ORG 31

▪ Lineweaver–Burk plot ▫ Based on Michaelis–Menten equation V [S] 1 K m + [S] → V0 = max = K m + [S] V Vmax [S] → 0 Figure 4.10 Processes that ↑ Vmax ↓ 1/Vmax → the line slopes lower on the graph than the control. Processes that ↓ Vmax ↑ 1/Vmax → the line slopes higher on the graph than the control. Figure 4.9 The Lineweaver–Burk plot shows Km and Vmax as functions of the x, y intercepts. AMINO ACID METABOLISM osms.it/amino-acid-metabolism ▪ Dietary protein broken down into amino acids → used to synthesize other proteins ▫ Excess amino acids used for energy/ stored as fat/glycogen ▪ Portal vein delivers absorbed amino acids (and other nutrients) to liver after uptake by small intestine → liver synthesizes needed proteins (e.g. albumin, immunoglobulins), non-essential amino acids ▪ Amino acids delivered to cells throughout body via blood → enter cell by facilitated/ active transport → used for protein synthesis (e.g. hormones, enzymes) ▪ Ammonia (NH4+): toxic metabolic byproduct from amino acid catabolism → converted to urea (liver) → eliminated (kidneys) Figure 4.11 Ammonia → urea in the liver. 32 OSMOSIS.ORG Transamination ▪ Reversible reaction ▫ Transfers nitrogen-containing amine group to another molecule ▪ Amino group transferred (via aminotransferase + vitamin B6 cofactor) ⇄ alpha ketoglutarate (acceptor molecule) → alpha-keto acid + glutamate ▫ Glutamate oxidatively deaminated in liver mitochondria → ammonia byproduct converted to urea (via urea cycle) → eliminated (kidneys) Deamination ▪ Nitrogen-containing amine group removal (via deaminase) → amino acid utilized for energy ▪ Produces ammonia → converted to urea → renal excretion

Chapter 4 Biochemistry: Protein Metabolism Figure 4.12 Example of a transamination reaction with amino acid alanine. ALT switches the amino group on alanine with the oxygen group on α-ketoglutarate, resulting in ketoacid pyruvate and amino acid glutamate, which has the amino group. Glutamate is the only amino acid that doesn’t have to transfer its amine group to another molecule. It undergoes oxidative deamination, a process that removes hydrogens and an amino group. NITROGEN & THE UREA CYCLE osms.it/nitrogen-and-urea-cycle ▪ Ammonia (NH3): toxic protein catabolism byproduct; detoxification by liver (forming non-toxic urea) Figure 4.13 Ammonia is composed of a nitrogen-containing amino group, an acidic carboxyl group, and a side chain. ▪ NH3 reaches liver in two ways, sometimes as glutamate Glutamine synthetase system ▪ From all tissues ▪ Glutamine synthetase: NH3 + glutamate → glutamine ▪ Glutamine transported through blood ▪ Glutaminase: glutamine → NH3 + glutamate ▪ In liver mitochondria Glucose-alanine cycle ▪ Only from muscle ▪ Glutamate dehydrogenase: NH3 + alphaketoglutarate → glutamate ▪ Alanine transaminase: glutamate + pyruvate → alpha-ketoglutarate + alanine ▪ Alanine transported through blood ▪ Alanine transaminase: alpha-ketoglutarate + alanine → glutamate + pyruvate Glutamate–NH3 conversion: two ways ▪ Glutamate dehydrogenase: glutamate → NH3 + alpha-ketoglutarate ▫ Free NH3 enters urea cycle ▪ Aspartate transaminase: glutamate + oxaloacetate → aspartate + alphaketoglutarate ▫ Aspartate carries NH3 into urea cycle OSMOSIS.ORG 33

Figure 4.14 The glutamine synthetase system of ammonia reaching the liver. 34 OSMOSIS.ORG

Chapter 4 Biochemistry: Protein Metabolism Figure 4.15 The glucose-alanine cycle of ammonia reaching the liver. OSMOSIS.ORG 35

Figure 4.16 Once glutamate is in a liver cell, there are two possible outcomes for it that depend on which enzyme it encounters (glutamate dehydrogenase or AST). In Option #1, ammonia enters the urea cycle; in Option #2, the ammonia group is carried into the urea cycle as part of the amino acid aspartate. Urea cycle ▪ Starts in liver cells’ mitochondria ▪ Carbamoyl phosphate synthetase 1 (CPS1) ▫ NH3 + CO2 + 2ATP → carbamoyl phosphate ▫ N-acetylglutamate → ↑ CPS1 affinity for ammonia (by allosteric binding) ▪ Ornithine transcarbamylase: ornithine + carbamoyl phosphate → citrulline + phosphate ▪ Citrulline moves to cytoplasm ▪ Argininosuccinate synthetase: citrulline + aspartate + ATP → argininosuccinate 36 OSMOSIS.ORG ▪ Argininosuccinate lyase: argininosuccinate → fumarate + arginine ▫ Fumarate → malate; malate → oxaloacetate (by malate dehydrogenase); oxaloacetate + glutamate → aspartate + alphaketoglutarate (by aspartate transaminase) → aspartate can enter next cycle ▫ Arginine → urea + ornithine (by arginase) → ornithine can enter next cycle ▪ Resulting urea then enters blood, excreted by kidneys

Chapter 4 Biochemistry: Protein Metabolism Figure 4.17 Illustration of the urea cycle, starting with the synthesis of carbamoyl phosphate from ATP, ammonia, and carbon dioxide, with the help of enzyme CPS1. OSMOSIS.ORG 37

PROTEIN STRUCTURE & SYNTHESIS osms.it/protein-structure-and-synthesis ▪ Proteins: functional structures composed of amino acids; synthesized within cells ▪ Genes, housed within DNA, provide blueprint for protein synthesis ▪ Codon: nucleotide triplet containing sequence of three nucleotide bases (A, G, T, C) ▫ Codes for specific amino acid ▫ 64 codons code for 20 amino acids; > one codon for most amino acids (UUU, UGC code cysteine) ▫ One “start” codon; three “stop” codons TRANSCRIPTION ▪ Messenger RNA (mRNA) transcribes code from DNA ▪ Begins at promoter ▫ Base sequence establishes transcription starting point Initiation ▪ RNA polymerase separates DNA helix at promoter site Elongation ▪ RNA polymerase unwinds, rewinds DNA → matches RNA nucleotides with DNA bases → links them together Figure 4.19 Elongation: RNA polymerase attaches complementary mRNA nucleotides to the unzipped DNA template strand to build an mRNA molecule. Termination ▪ Ends at termination signal; base sequence establishes transcription end point Figure 4.18 The four nucleobases used in DNA are guanine, cytosine, thymine, and adenine. In mRNA, uracil (U) is used rather than thymine. 38 OSMOSIS.ORG Pre-mRNA formed ▪ Contains non-coding areas (introns) ▫ Spliceosomes snip out introns → functional mRNA ▫ mRNA complex proteins added → guide mRNA out of nucleus

Chapter 4 Biochemistry: Protein Metabolism Figure 4.20 Termination: when the two complementary sequences in the terminator sequence get transcribed into mRNA, they bond with each other, creating a hairpin loop that causes the RNA polymerase to detach from the DNA strand. TRANSLATION ▪ Base sequence contained in mRNA translated into assembled polypeptide Three RNA types required ▪ mRNA: carries coded message out of nucleus to ribosome in cytoplasm ▪ Ribosomal RNA (rRNA): “workbench” for protein synthesis ▪ Transfer RNA (tRNA): brings amino acids to workbench assembly site at ribosome ▫ Folded into “cloverleaf” shape ▫ Acceptor stem: attaches to amino acid ▫ Anticodon: complementary to mRNA codon (tRNA binds with mRNA through complementary base pairing) OSMOSIS.ORG 39

Figure 4.21 Translation: as ribosomes line tRNA molecules up with their complementary codons, the amino acids held by the tRNA bind with each other to form a protein, which is a chain of amino acids. The process is terminated at a stop codon. 40 OSMOSIS.ORG