Myoglobin and Hemoglobin

• Globins are oxygen-binding proteins like myoglobin and hemoglobin.

Myoglobin

• A muscle protein.
• It is a heme-containing monomeric protein.
• Its major function is to store O2.

Hemoglobin

• A major component of red blood cells.
• It is a heme-containing tetramer composed of 4 polypeptide chains (2 alpha + 2 beta).
• Its major function is to carry O2 from the lungs to the tissues.

Structure

• Myoglobin is primarily an alpha-helical structure with turns and loops.
• Water-soluble globular proteins form complex 3D structures, containing a heme group and an iron atom.
• Heme structure contains a protoporphyrin and a central iron atom in the ferrous (Fe2+) form.
• Iron lies in the center of protoporphyrin and has 6 coordination bonds: 4 in the plane of the porphyrin ring bound to nitrogens, and two perpendicular (fifth and sixth coordination sites).
• The fifth site is occupied by an imidazole ring of a histidine, known as the proximal histidine.
• The sixth site binds oxygen.
• Upon oxygen binding, the iron moves into the plane of the protoporphyrin ring.
• The iron lies slightly outside the plane of porphyrin in deoxyhemoglobin, but upon oxygenation, it moves into the plane of the heme.
• Hemoglobin is a tetramer of two identical dimers; its formula is α2β2.
• Hemoglobin (beta) subunits are structurally similar to myoglobin.

Oxygen Binding

• The Y-axis represents the saturation of heme binding by myoglobin and hemoglobin for each O2.
• Higher concentrations show more efficient binding of oxygen.
• To change the affinity for oxygen, the protein must have multiple binding sites that can interact with each other (cooperativity).
• Positive cooperativity: the first binding event increases affinity at remaining sites, recognized by sigmoidal binding curves.
• Negative cooperativity: the first binding event decreases affinity at remaining sites.
• T (tense) state: more interactions/more stability and a lower affinity for O2.
• R (relaxed) state: fewer interactions/more flexibility and higher affinity for O2.
• O2 binding promotes a T → R conformational change.
• An allosteric protein is one where the binding of a ligand to one site can affect the binding properties of another site on the same protein; it can be positive or negative.
• Homotropic: the normal ligand of the protein is the allosteric regulator.
• Heterotropic: a different ligand affects the binding of the normal ligand.
• Cooperativity = positive homotropic regulation.
• More BPG results in easier release/attraction of (?) oxygen.

2,3-Biphosphoglycerate

• Regulates O2 binding.
• It is a negative heterotropic regulator of hemoglobin function.
• It is produced from an intermediate in glycolysis.
• A small negatively charged molecule binds to the positively charged central cavity of hemoglobin and stabilizes the T state.
• It is a byproduct of glycolysis.

Carbon Dioxide

• Produced by metabolism in tissues, binds to hemoglobin (not heme) and stabilizes the T state, helping to release oxygen in tissues.
• Transport by Hb accounts for 14% of the total CO2 transport; it is transported through blood as bicarbonate formed spontaneously or through the action of the enzyme carbonic anhydrase.
• Tissues actively metabolize, generating H+, which lowers the pH of the blood near the tissues relative to the lungs (catalyzed by carbonic anhydrase).
• H+ binds to Hb, stabilizes the T state, and leads to the release of O2 in the tissues (Bohr effect).
• In equilibrium, protons, CO2, and 2,3-BPG push the reaction towards the T state to compete with oxygen binding.

Enzyme Action, Kinetics, and Regulation

Enzymes

• Catalysts that increase reaction rates without being used up.
• Most are globular proteins, but some RNAs (ribozymes and rRNA) also catalyze reactions.
• Carbonic anhydrase catalyzes a hydration reaction between CO2and H2O to produce H2CO3as an intermediate and H+ and HCO3- as the final product.
• CO2 + H2O → H2CO3 → H+ + HCO3-
• As one of the fastest enzymes, it can hydrate 10^6 molecules of CO2 per second; this catalyzed reaction is 10^7 times as fast as the uncatalyzed one.

Enzyme Specificity

• Reactants in enzyme-catalyzed reactions are called substrates.
• Proteolytic enzymes (proteases) catalyze the hydrolysis of peptide bonds.
• Papain from papaya plants cleaves any peptide bonds, exhibiting broad specificity.
• Trypsin cleaves the carboxyl side of Arg and Lys residues.
• Thrombin cleaves Arg-Gly bonds in select sequences (thrombin has very high specificity) and is important for blood clotting.

Six Major Classes of Enzymes:

• Oxidoreductases: involved in oxidation and reduction.
• Transferases: transfer functional groups between molecules.
• Hydrolases: cleave molecules by adding water (catalyze hydrolysis of a substrate).
• Lyases: add atoms or functional groups to a double bond or remove them to form double bonds.
• Isomerases: catalyze rearrangements of atoms within a molecule.
• Ligases: join two molecules.

Enzyme Activation

• Requires cofactors: metal ions or coenzymes (organic molecules).
• Coenzymes contain cosubstrates (transiently associated with the enzyme, like NADH) or prosthetic groups (permanently associated with the enzyme and usually covalently bound, like heme, FADH2).
• An enzyme with its cofactor is a holoenzyme; without a cofactor, it’s an apoenzyme.
• Tightly bound coenzymes are called prosthetic groups.
• Enzymes do not alter the ΔG of a reaction (change of energy in a reaction: measure of useful energy).
• They do not affect equilibrium, meaning they cannot affect the free energy of the reaction.
• Slow reactions face significant activation barriers that must be surmounted during the reaction.
• Enzymes increase reaction rates by decreasing the free energy of activation.
• Enzyme-catalyzed reactions take place in the active site.
• The molecule that is bound to the active site is called the substrate, thus forming the enzyme-substrate complex and drives selectivity.
• Interactions between enzyme and substrate promote the formation of the transition state.
• Active sites may include distant residues.
• Induced fit: enzymes change shape upon substrate binding; the active site forms a shape complementary to the substrate after the substrate has been bound, transiently changing the enzyme.

Kinetics of Enzyme-Catalyzed Reactions

• Parameters to characterize include Vmax (maximum velocity), Kcat (turnover number), and Km (Michaelis constant).
• The enzymatic reaction reaches Vmax when the enzyme is saturated with the substrate.
• Vmax varies with the amount of enzyme in the reaction; reached when the enzyme is saturated with substrate.
• Vmax is dependent on the amount of enzyme in a reaction.
• Kcat = Vmax / [enzyme]. Kcat- is a turnover number that describes the number of substrate molecules converted to a product in a given unit of time in one enzyme molecule
• It measures the velocity independent of the concentration of the enzyme and is a constant for the enzyme under a given condition.
• The ratio stays constant per active site.
• Km is the Michaelis constant for an enzyme and describes the required substrate concentration to get Vmax/2.
• It measures the enzyme’s affinity for the substrate but is inversely related to affinity– higher Km, lower affinity for the substrate.
• A lower concentration of the substrate can demonstrate a higher affinity of the enzyme.
• Plotting 1/v0 vs 1/[S] will create a straight line.
• Km is a negative reciprocal of the x-intercept, and Vmax is an inverse of the y-intercept.
• Enzymes are modulated by temperature, pH, and inhibitors.
• Interaction with the substrate depends on the shape of the enzyme, and the shape of the enzyme is affected by temperature, pH, and certain molecules.

Influence of Temperature and pH

• Enzyme activity increases with temperature until it denatures.
• Optimal pH for pepsin: 1.5; optimal pH for chymotrypsin: 8.5.
• Certain ionizable R groups affect the pH dependence of enzymes.

Inhibitors

• Decrease enzyme activity.
• Irreversible (inactivators): react with enzymes and can permanently turn off enzymes; usually powerful toxins and can be used as drugs; can be caused by covalent modification of a crucial residue at the active site.
• Reversible inhibitors: bind to the enzyme and then dissociate; structural analogs of substrates and used as drugs to slow down certain enzymes; can bind to a free enzyme and prevent a substrate from binding, and to an enzyme-substrate complex and prevent the reaction from taking place.
  • Competitive: takes the place of where the substrate should be by resembling the substrate; replaces the substrate at the active site; does not change Vmax; Km increases and the inhibition can be overcome by a higher amount of substrate concentration.
  • Uncompetitive: binds to the E-S complex; does not affect substrate binding and inhibits the catalytic function; decreases both Vmax and Km; the ratio of Km/Vmax does not change; on a Lineweaver-Burk plot, both lines are parallel.
  • Noncompetitive: binds to the regulatory site, regardless if there is a substrate or not; changes the shape of the active site, so the substrate will not fit properly; decreases Vmax but does not change Km (allosteric).

DNA Replication

• Central dogma of biology: DNA → transcription → RNA → translation → protein; DNA has the ability to replicate itself.
• There are around 4.6 million E. coli nucleotides; 3 billion in the human genome.
• For synthesis to begin, DNA polymerase requires a primer; the polymerase can only extend in the 5' → 3' direction.
• RNA primers are replaced with DNA nucleotides, and the fragments must be joined.
• Semiconservative mechanism of replication (Watson and Crick): two new molecules are produced, each with one new strand and one conserved (old) strand.
• Replication is initiated at sites called origins of replication and usually proceeds bidirectionally.
• For circular (plasmids, plastids, mitochondria, bacteria/prokaryotes) DNA, there is a single origin of replication.
• There are 46 molecules of DNA (in humans).
• The double-helical parental molecule serves as a template.
• Helicases use ATP hydrolysis to separate the strands of the helix to make the DNA available for the polymerase.
• The helicase consists of a ring-like structure composed of six subunits to pry the helix apart.
• Unwinding puts a strain on the molecule caused by the topological stress (overwinding) of nearby regions.
• Topoisomerases relieve the stress by cutting the DNA and allowing the strands to swivel around each other to release the tension before rejoining.
• DNA polymerase can’t begin synthesis de novo; RNA polymerase can begin synthesis de novo; it can only add new nucleotides to the 3' end of the existing chain.
• Primase synthesizes short RNA (=10 nucs) complementary to the DNA strand that serves as a primer for synthesis; the primer is removed later.

Key Characteristics of DNA Synthesis

• Four deoxynucleoside triphosphates and Mg2+ are required.
• The template strand directs synthesis.
• A primer for the new strand must be present.
• Polymerases have nuclease activity that allows for the removal of mismatched bases (on ends).
• Nucleoside triphosphates act as substrates during synthesis.
• Pyrophosphate (made of beta and gamma phosphates) is a good leaving group.

RNA Synthesis, Processing, and Regulation

• Ribosomes are next to DNA.
• There is no mRNA processing in a prokaryotic cell.
• Transcription and replication chemical mechanisms both require Mg2+ ions and nucleoside triphosphates (NTPs); they both synthesize 5' → 3', and the DNA template is required.
• Transcription only involves 17 base pairs of DNA (being unwound) at a given time.
• Only one strand works as a template, and primers are not required.

Products of RNA Copies

• RNA can be either coding (mRNA) or noncoding (rRNA, tRNA, siRNA, miRNA, etc.).
• The fraction of genomic DNA corresponding to transcribed protein-coding genes is inversely related to genome size and the amount of DNA transcribed into noncoding RNA.
• Prokaryotes have one RNA polymerase, while eukaryotes have multiple:
  • RNA polymerase I: synthesizes preribosomal RNA (precursor for 28S, 18S, and 5.8S rRNAs transcribed as a single transcript); in nuclei.
  • RNA polymerase II: synthesis of mRNA; very fast (500-1000 nucs/sec), recognizes thousands of promoters; requires proteins for activity called transcription factors.
  • RNA polymerase III: makes tRNAs and some small RNA products.
  • Plants have polymerase IV that synthesizes small interfering RNAs; mitochondria have their own polymerase.
• DNA template serves as a template for RNA polymerase, and DNA coding is the non-template that has the same sequence as the RNA transcript (only thymine is replaced by uracil); the TEMPLATE IS WHAT IS USED; regulatory sequences are listed by the coding strand sequence.
• The promoter is a specific DNA sequence where RNA polymerase binds.
• In E. coli, there is the -10 sequence (Pribnow box) and the -35 sequence as promoters.
  • TATAAT: average or consensus sequence.
• Transcription bubble: region containing RNA polymerase, DNA, and the RNA product.
• Unprocessed RNA is considered a primary transcript; the majority of RNA molecules are processed after synthesis.
• Eukaryotic mRNA and tRNA (prokaryotic and eukaryotic) are the most extensively processed.
• Processing of eukaryotic mRNA includes adding a 5' cap, a 3' poly(A) tail, and splicing out introns and ligating exons.

mRNA Processing

• 7-methylguanosine links to the 5' end via a 5',5'-triphosphate link; formed with a molecule of GTP; includes additional methylations at the 2'OH groups of the next two nucleotides after the cap; protects RNA from nucleases; forms a binding site for the ribosome.
• Introns are the part of the nucleotide sequence that is removed during maturation of the final RNA product; exons are less than a thousand bp in length, while introns are between 50-70000 bp in length (usually around 1800).
• The human genome has more than 200,000 introns spread across 20,000 genes.
• Four classes of introns based on how they are spliced: group 1 and group 2 are self-splicing, spliceosome introns are spliced by the spliceosome, and tRNA introns are spliced by protein-based enzymes.

Genetic Code

• A four-letter code in groups of two amino acids is insufficient (16 combinations), and a four-letter code in groups of three amino acids is sufficient (64 combinations).
• Living organisms use nonoverlapping mRNA code with no punctuation.
• A group of three nucleotides is called a codon.
• The genetic code is universal.
• 61 codons code for amino acids; three are terminations (UAA, UGA, UAG) and one is an initiation code (AUG/methionine codon).
• It is written in the 5' → 3' direction.
• The third base is less important in binding to tRNA, while the first codon establishes the reading frame (if the frame is thrown off by a single base, then all subsequent codons are messed up).
• There are multiple codons that code for each amino acid, except methionine (start codon) and tryptophan.
• Proteins have the lowest concentrations of Met and Trp.
• The third base is not as important because there are usually multiple ways to code for the same amino acid– where the third base is different.
• When there is a change in the third base, but no difference in the amino acid that it codes, that causes a silent mutation.
• tRNAs are the adaptors that recognize codons and insert anticodons (the adaptor) that are attached to amino acids into their respective places.
• The codon in mRNA base pairs with the anticodon in tRNA through hydrogen bonding.
• This creates an antiparallel alignment of the two RNA segments.
• The amino acid is coded by the mRNA codon, not the tRNA anticodon.

tRNA

• Usually between 73-93 nucleotides in length, and the 3D molecule is an L-shape.
• The 3' CCA terminal region is where the amino acid is accepted and it is called the acceptor stem.
• Activated amino acids are attached to a hydroxyl group of adenosine in the CCA region of the acceptor stem.
• The 5' end is phosphorylated, and the 5' terminal residue is usually pG.
• The anticodon is located in one of the loops of the tRNA molecule, near the center.
• tRNA molecules contain unusual bases like inosine or bases that have been modified.
• Inosine is formed by deamination of adenosine after the synthesis of the primary transcript.
• tRNA molecules can recognize more than one codon because of wobble base pairing (pairing among RNA molecules that do not follow the standard Watson-Crick base pairing).

Five Stages of Protein Synthesis:

• Activation of amino acids (tRNA aminoacylated).
• Initiation of translation (mRNA and aminoacylated tRNA bind to the ribosome).
• Elongation (cycles of aminoacyl-tRNA binding and peptide bond formation until a stop codon is reached).
• Termination and ribosome recycling (mRNA and protein dissociate, and the ribosome is recycled).
• Folding and posttranslational processing (catalyzed by a variety of enzymes).
• Amino acids are activated when an ester linkage between the carboxyl group of the amino acid and either the 2' or 3' hydroxyl group of the terminal adenosine of the tRNA is formed → aminoacyl tRNA or charged tRNA.

Aminoacyl tRNA Synthetases Catalyze the Activation of Amino Acids

• Aminoacyl tRNA synthetases translate the genetic code by assigning a certain amino acid to its respective tRNA; they recognize the anticodon loops and acceptor stems of tRNAs.
• They have high discriminatory characteristics regarding amino acid activation sites.
• Each aminoacyl tRNA synthetase is specific for a particular amino acid.
• Threonyl tRNA synthetase has a zinc ion at its active site, which interacts with the hydroxyl group of threonine.
• Valine is structurally similar to threonine, but it lacks the hydroxyl group and is not joined to the tRNA(Thr).
• Serine, smaller than threonine, is occasionally linked to tRNA(Thr) because of the presence of its hydroxyl group.
• Editing of aminoacyl tRNA is critical for ensuring fidelity.

Mechanism of Protein Synthesis

• Ribosome = ribonucleoprotein (large subunit + small subunit).
• E. coli ribosome settles at 70S; contains a large 50S subunit and a small 30S subunit.
• 50S is composed of 34 proteins and a 23S rRNA and 5S rRNA.
• 30S is composed of 21 proteins and 16S rRNA.
• ⅔ of the ribosome’s mass is used for RNA (structure and function).
• rRNA is the catalyst for protein synthesis; ribosomal proteins have a small contribution.

Eukaryotic vs. Bacterial Ribosome Composition

• Eukaryotic Ribosome:*
  • Ribosome sedimentation coefficient: 80S
  • Large subunit sedimentation coefficient: 60S
  • Large subunit rRNAs: 28S rRNA, 5S rRNA, 5.8S rRNA
  • Small subunit sedimentation coefficient: 40S
  • Small subunit rRNA: 18S rRNA
• Bacterial Ribosome:*
  • Ribosome sedimentation coefficient: 70S
  • Large subunit sedimentation coefficient: 50S
  • Large subunit rRNAs: 23S rRNA, 5S rRNA
  • Small subunit sedimentation coefficient: 30S
  • Small subunit rRNA: 16S rRNA

Ribosomal tRNA Binding Sites:

• A (aminoacyl) site: binds incoming tRNA.
• P (peptidyl) site: binds tRNA with the growing peptide chain.
• E (exit) site: binds uncharged tRNA before it leaves the ribosome.
• The large subunit is responsible for peptide bonding; the small subunit decodes genetic information.
• The ribosome moves toward the "right" side of mRNA to read the strand from 5' → 3'.

Five Stages of Protein Synthesis:

• Activate amino acids (tRNA aminoacylated).
• Initiate translation (mRNA and aminoacylated tRNA bind to the ribosome).
• Elongate (cycles of aminoacyl-tRNA binding and peptide bond formation until a stop codon is reached).
• Terminate and recycle the ribosome (mRNA and protein dissociate, and the ribosome is recycled).
• Folding and posttranslational processing (catalyzed by a variety of enzymes).

Initiation

• When mRNA is polycistronic, that means it codes for more than one protein (bacterial mRNA molecules are polycistronic).
• Formylmethionyl tRNA initiates bacterial protein synthesis.
• Formylmethionyl is a modified form of methionine; N-formylmethionine (fMet) is the initiator amino acid in most bacterial proteins.
• tRNA(f) (initiator tRNA) with fMet-tRNA(f) binds to the initiation codon (AUG) once; does not bind to other AUG codons.
• tRNA(m) recognizes internal codons for methionine.
• The same synthetase activates both tRNA(m) and tRNA(f), and a specific transformylase modifies the methionine attached to tRNA(f).
• Initiation factors aid in the assembly of the 30S initiation complex and then the 70S initiation complex.
• Formylmethionyl-tRNA(f) is placed in the P site of the ribosome in the formation of the 70S initiation complex (formation of this complex is a rate-limiting step in protein synthesis).

Elongation

• Aminoacyl tRNA is delivered to the A site of the ribosome in association with a protein called elongation factor Tu (EF-Tu); EF-Tu delivers tRNA to the A site on the ribosome.
• Process: aminoacyl-tRNA binding → peptide bond formation → translocation/elongation factor G (GDP+Pi) → tRNA dissociation.

Termination

• A release factor recognizes a stop codon in the A site and stimulates the release of the completed protein from the tRNA in the P site.

Eukaryotic Translation Initiation

• Starts with the assembly of a complex on the 5' cap that includes the 40S subunit and Met-tRNA(i).
• This complex scans mRNA using energy from ATP hydrolysis until the first AUG is reached.
• The 60S subunit is added to form the 80S initiation complex.
• Streptomycin interferes with the binding of fMet-tRNA and inhibits protein synthesis initiation in bacteria.
• Puromycin inhibits protein synthesis in eukaryotes and bacteria by releasing uncompleted polypeptide chains.