Analytical Ultracentrifugation Techniques
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Questions and Answers

What type of sedimentation technique involves using very fast rotor speeds, leading to sedimentation velocity without back-diffusion of protein solute?

  • Non-equilibrium sedimentation (correct)
  • Fluorescence sedimentation
  • Equilibrium sedimentation
  • Refractive index sedimentation

    • Which force is NOT experienced by solute molecules during sedimentation in analytical ultracentrifugation (AUC)?

  • Frictional force
  • Magnetic force (correct)
  • Buoyancy force
  • Centrifugal force

    • In equilibrium sedimentation, what happens to protein sedimentation once equilibrium is reached?

  • Only the monomer form of protein exists
  • Protein diffuses completely out of the solution
  • Protein concentration decreases
  • Sedimentation is balanced by diffusion back up the cell (correct)

    • What is the primary method that has now mostly superseded equilibrium sedimentation for estimating molecular weight?

    <p>Mass spectrometry</p> Signup and view all the answers

    Which detection method in analytical ultracentrifugation is considered to be universal and does not require absorbing protein?

    <p>Refractive index detection</p> Signup and view all the answers

    Which amino acid forms the nucleophilic attack in papain?

    <p>Cysteine</p> Signup and view all the answers

    What is the role of Histidine in the catalytic dyad of papain?

    <p>General acid/base catalyst</p> Signup and view all the answers

    Which of the following is NOT a function of metal ions in the human body?

    <p>Act as nucleophiles in enzymatic reactions</p> Signup and view all the answers

    What is an example of aspartyl protease?

    <p>Chymosin</p> Signup and view all the answers

    Which statement regarding thiol proteases is accurate?

    <p>They use a C, H catalytic dyad.</p> Signup and view all the answers

    Which metals are typically abundant in the human body?

    <p>Calcium, Magnesium, Sodium</p> Signup and view all the answers

    What role do coordinating residues in proteins play regarding metal ions?

    <p>Bind to metal ions</p> Signup and view all the answers

    What is a characteristic of porphyrins?

    <p>They act as chelating ligands with multiple donor atoms.</p> Signup and view all the answers

    What type of ligands coordinate with Cu in Type II copper sites?

    <p>Two Histidines and one Cysteine</p> Signup and view all the answers

    What is a characteristic of Type III copper centers?

    <p>They are EPR silent due to antiferromagnetic coupling</p> Signup and view all the answers

    What is the role of LPMOs in enzymatic processes?

    <p>Degrading polysaccharides</p> Signup and view all the answers

    How do LPMOs activate molecular oxygen?

    <p>Triggered by binding of the substrate</p> Signup and view all the answers

    What reaction do galactose oxidases catalyze?

    <p>Oxidation of alcohols to aldehydes</p> Signup and view all the answers

    What type of geometry is exhibited by the copper in galactose oxidases?

    <p>Distorted square pyramidal</p> Signup and view all the answers

    What is one suspected role of galactose oxidases?

    <p>Antibacterial production of H2O2</p> Signup and view all the answers

    Which amino acid plays a crucial role in the unique active site of LPMOs?

    <p>Histidine</p> Signup and view all the answers

    What role does Zn play in the catalytic mechanism of carboxypeptidase A?

    <p>Polarizes the C=O bond as a Lewis acid</p> Signup and view all the answers

    Which residue facilitates the addition of a proton to the amide during the reaction catalyzed by carboxypeptidase A?

    <p>Tyr</p> Signup and view all the answers

    What is the effect of Zn removal from carbonic anhydrases?

    <p>Loss of activity but no structural change</p> Signup and view all the answers

    Which mechanism describes the action of carbonic anhydrase in the hydration of CO2?

    <p>Nucleophilic attack by hydroxide ion on CO<del>2</del></p> Signup and view all the answers

    What is the primary influence of Zn in the mechanism involving water and the C=O bond?

    <p>Lowers the pK<del>a</del> of bound water, forming hydroxide</p> Signup and view all the answers

    What is the main role of carbonic anhydrases in the human body?

    <p>Catalyzing the interconversion of CO<del>2</del> and carbonic acid</p> Signup and view all the answers

    What is the consequence of protonation and deprotonation in the catalytic cycle of carbonic anhydrase?

    <p>Facilitates the closure of the catalytic cycle</p> Signup and view all the answers

    Which disease is associated with changes in iron levels in the human body?

    <p>Anaemia</p> Signup and view all the answers

    What is the role of NAD+ in enzymatic reactions?

    <p>Accepts hydrogen from alcohol during oxidation</p> Signup and view all the answers

    What characterizes an enzyme that is termed as an apoenzyme?

    <p>It lacks its required cofactor</p> Signup and view all the answers

    Which vitamin is the precursor for pyridoxal phosphate (PLP)?

    <p>Vitamin B6</p> Signup and view all the answers

    How do exopeptidases and endopeptidases differ in their function?

    <p>Exopeptidases cleave terminal bonds; endopeptidases cleave internal bonds</p> Signup and view all the answers

    What is the active site composition of serine proteases?

    <p>Aspartate, histidine, and serine</p> Signup and view all the answers

    Which factor influences the specificity of trypsin among serine proteases?

    <p>Positive charge on the amino acid</p> Signup and view all the answers

    What type of reaction do serine proteases primarily facilitate?

    <p>Hydrolytic reactions</p> Signup and view all the answers

    What is a key feature of cobalamin (Vitamin B12)?

    <p>It shuttles between three different oxidation states</p> Signup and view all the answers

    What is the primary function of CYP450 monooxygenases in the human liver?

    <p>Specific oxidation during biosynthesis or biodegradation</p> Signup and view all the answers

    Which phase in the mechanism of CYP450 involves changing Fe from low spin to high spin?

    <p>Substrate binding</p> Signup and view all the answers

    How does the P450 enzyme activate molecular oxygen?

    <p>Through substrate binding triggering a conformational change</p> Signup and view all the answers

    What is the result of the reaction where P450 monooxygenases perform hydroxylation of substrates?

    <p>Insertion of an oxygen atom into the substrate</p> Signup and view all the answers

    What type of ligands does Cu(I) prefer for coordination?

    <p>Softer donors such as sulfur or phosphorus</p> Signup and view all the answers

    Which characteristic defines Type I Cu-proteins?

    <p>Mononuclear sites with high reduction potential</p> Signup and view all the answers

    What type of oxygen transport protein is hemocyanin?

    <p>Copper-based</p> Signup and view all the answers

    What happens to Fe during the catalytic cycle of P450 enzymes?

    <p>It oscillates between Fe(II), Fe(III), and Fe(IV) oxidation states</p> Signup and view all the answers

    Signup and view all the answers

    Study Notes

    Enzymology - Lecture 1

    • Evolution is not about survival of the fittest, but the death of the unfit. Evolution changes at the DNA level, which changes the phenotype of an organism. Evolution is a change in proteins.
    • Random mutations are single base changes
    • Genetic drift is the accumulation of these changes in a population
    • Selection removes unfavorable changes. Without selection, genetic drift is random.
    • Divergent evolution is one species splitting into multiple separate species, without cross-breeding.
    • Convergent evolution is where enzymes have similar function but different structures.
    • EC numbers describe the function of an enzyme.
    • Primary structure is the assembly of amino acids.
    • Secondary structure is the folding of amino acids.
    • Tertiary structure is the packing of the amino acid chain.
    • Quaternary structure is the interaction of the amino acid chains.
    • All amino acids have a chiral alpha carbon, bonded to four different groups.
    • The 'CORN' rule determines the L or D configuration of the molecule (clockwise/anti-clockwise rule of naming).
    • Follow all one letter and three letter codes, features (size, charge at physiological pH), and examples provided.

    Amino Acids

    • All amino acids in list, one-letter and three-letter codes, and features are included.

    Lecture 2

    • Hydrogen bonds between the main chain N and O atoms are important for helix stabilisation.
    • 3.6 amino acids per complete turn of alpha helix.
    • Every 7th amino acid is next to each other in an alpha helix.
    • Side chains stick out of α-helix, giving different chemistry to opposite sides.
    • Hydrogen bonds between main chain N and O atoms determine the sheet stabilisation in beta pleated sheets and sheets can be parallel or anti-parallel.
    • Side chains stick out from β-sheets.

    Lecture 3

    • All enzymes within the same family have the same catalytic residues.
    • Enzymes can change shape (e.g. by binding a substrate).
    • Allosterics: This is where the binding of one substrate affects the binding of another. Haemoglobin is an example.
    • Enzyme Commission (EC) numbers describe an enzyme. They have a 4-digit number to describe the enzyme class, the bond acted on, the donor, and the acceptor of the reaction acted on.
    • Rate of reaction (Vo), Vmax, KM, kcat
    • All about Enzyme Kinetics, rate constants, including the Lineweaver–Burk plot.
    • Reversible inhibitors can be removed, and the enzyme will recover.
    • Non-reversible inhibitors require chemical reaction to remove.
    • All detail about inhibitors including competitive and non-competitive inhibitors.
    • Cofactors are Chemical compounds required for an enzyme's function
    • Coenzymes are functional compounds required by enzymes.
    • Details about cofactors and coenzymes including NAD in the text.
    • Prosthetic groups are often involved in structural stability.
    • Details on Glycoproteins and Glycosylations (N-Glycans and O-Glycans and their importance in protein folding). Includes details on their role in cell-cell recognition.

    Lecture 4

    • Details on Vitamins (Biotin, Thiamine pyrophosphate (TPP) and Cobalamin).

    Other Topics

    • Details on proteins, Protein structures (clustering of polypeptide chains), and quaternary structures.
    • All detail provided concerning Metal Ions (including their coordination, Coordination modes, and their role in metalloenzymes; e.g. Zinc and its role in carbonic anhydrase, carboxypeptidase, and superoxide dismutase (SOD).)
    • Details on Iron including its role in ferritin, transferrins, and Fe based proteins in respiration.
    • Details on Nitrogen Radicals and their impact on O2 activation reactions, Cytochrome P450 (CYP450). Details on specific enzymes e.g. Superoxide dismutase, catalase and glutathione peroxidase. Details on different states of Cu ions and proteins.

    Lecture 5

    • Details on Galactose oxidases and Cu(I)/Cu(II) in different proteins. Details are given regarding Cu role in Monooxygenases, and Cu and the reactions with enzymes including Cytochrome P450 proteins. Details on how the reaction proceeds to regenerate to the initial state.

    Other

    • Further Detail on protein and enzyme assays and methods (including methods to separate and study proteins like: NMR, PAGE, IEF, SDS-PAGE). Also, detail on the use of different techniques used to monitor protein folding, and protein-ligand interactions. Details are given concerning techniques to monitor protein sequencing, including specific mentions of trypsin usage and products in this process.
    • Specific mention of different experimental techniques including UV-absorption, Fluorescence, and Sedimentation, including a detailed explanation of each technique and its practical advantages and limitations. This will include explanations of each technique within biological contexts.
    • Detail of the different kinds of proteins used for binding and/or transport (e.g Lysozyme, and haemoglobin, and their functions).
    • Details on plasmids, DNA ligases, PCR and cell transformation techniques and mechanisms.

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    CHEM3012 Enzymology Lecture 1 PDF

    Description

    Test your knowledge on analytical ultracentrifugation and its techniques, including sedimentation velocity, equilibrium sedimentation, and the role of metal ions in proteins. This quiz covers essential concepts and methods used in protein analysis.

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