UWorld Biochemistry: Enzyme Quiz #2

• Catalytic efficiency indicates enzyme effectiveness when substrate concentration is low.
• Higher catalytic efficiency arises in enzymes that bind substrate well (lower KM), irrespective of a slower rate (lower kcat).
• Catalytic efficiency is determined by Kcat/km, not just kcat.
• Low kcat values can still be efficient, provided there is a significant drop in KM.
• At low substrate concentrations, catalytic efficiency becomes the primary determinant.
• Phosphorylation is a covalent modification, not allosteric regulation.
• Allosteric regulation involves noncovalent, reversible interactions that are usually ligand-based.
• Activation energy (Ea) equals the energy of the transition state minus the energy of the reactants.
• Catalytic turnover is represented by kcat and is proportional to the maximum reaction velocity, which is obtained at high substrate concentrations.
• Catalytic efficiency is represented by kcat/Km and reflects the competence of the enzyme at low substrate concentrations.
• Kcat= vmax/E, where E = enzyme concentration

Enzymes and Energy

• Enzymes reduce Ea, without affecting ∆G or Keq.
• Active site mutations can increase Kd and reduce the reaction rate.
• ∆G and Keq are solely dependent on reactants and products, and are not influenced by enzymes.
• Enzymes do not change the equilibrium constant (Keq) or the overall free energy change (ΔG) of a reaction.
• The transition state is always higher in energy than both the reactants and the products, even when stabilized by an enzyme.
• Enzymes lower activation energy by stabilizing the transition state, speeding up both the forward and reverse reactions.
• Enzymes lower Ea, but they do not affect ∆G (Gibbs free energy) of the reaction.

Proteases

• Protease activity relies on hydrolysis, where water is utilized to cleave peptide (amide) bonds.
• Proteases are enzymes that specifically break peptide bonds in proteins.
• Cleavage only occurs when a protease recognizes and acts on its specific substrate.
• Proteases are hydrolases that break peptide bonds using water (hydrolysis)
• Secreted proteases are active outside the cell in the extracellular space.

Gibbs Free Energy

• ∆G < 0: Spontaneous, exergonic, and energy-releasing reactions.
• ∆G > 0: Nonspontaneous, endergonic, and energy-requiring reactions.
• ∆G = Gproducts − Greactants : Calculation of Gibbs free energy.

Protein Folding

• Protein folding raises the entropy (∆S) of water, since hydrophobic residues disrupt rigid solvation layers, and increase water disorder.
• The entropy (∆S) becomes positive, and water molecules gain freedom upon protein folding.
• Enzymes act as catalysts by lowering activation energy, accelerating reactions without being permanently changed.

Kinetics

• An increase in enzyme concentration or kcat results in an elevated Vmax
• Altering enzyme concentration affects Vmax, without changing KM
• KM is a reflection of substrate binding affinity instead of enzyme quantity
• Y-intercept decreases when Vmax increases.
• X-intercept remains constant when KM is unchanged.
• Product formation being the rate-limiting step means kcat is small, since kcat measures how fast an enzyme converts substrate to product.
• Fluorescence intensity is a direct measure of product formation (more fluorescence = faster catalysis = higher kcat and/or higher enzyme concentration).
• If a substrate is still cleaved after knocking out an enzyme, it implicates low specificity and cleavage by other enzymes. Enzymes with low specificity can act on multiple different substrates.
• A lower KM shifts the x-intercept left (further from zero).
• A higher Vmax moves the y-intercept closer to zero.
• A lower KM and/or higher Vmax results in a smaller slope on graphs.
• A higher KM and/or lower Vmax results in a larger slope on graphs.
• If V₀ is close to Vmax, then [S] must be greater than KM in Michaelis-Menten problems.
• If V₀ is much less than Vmax, then [S] is likely near or below KM in Michaelis-Menten problems.
• When V₀ is given as a percent of Vmax, substrate concentration [S] can be estimated as a multiple of KM.
  • If V₀ = 75% of Vmax, then [S] ≈ 3 × KM.
• In Lineweaver-Burk plots, the solid line is usually the reference (normal enzyme).
• Changes in dashed lines reflect experimental conditions.
• If the slope on a Lineweaver-Burk plot decreases, Vmax increased (or KM decreased).
• If the slope increases, Vmax decreased (or KM increased) on Lineweaver-Burk plot.
• Match substrate concentration to KM to test inhibitor effectiveness under realistic conditions.
• Enzymes lower the activation energy to speed up the reaction but do NOT change the Gibbs free energy (ΔG).

Enzymes and Redox

• Disulfide bond cleavage is a reduction reaction, catalyzed by oxidoreductases.
• Isomerases catalyze atomic rearrangement and cannot cleave disulfide bonds.
• Phosphorylases use inorganic phosphate to break bonds and do not act on disulfide bonds.
• Reactions involving breaking or forming disulfide bonds are redox reactions, catalyzed by oxidoreductases.
• Dehydrogenases are oxidoreductases that move electrons, not phosphates.

Hypothesis Testing

• Rejecting a false null hypothesis based on a significant p-value (≤ 0.05) supports the alternative hypothesis.
• Type I error represents a false positive.
• Type II error represents a false negative.
• Failing to reject a true null hypothesis is the correct decision.

Enzymes

• For an enzyme inhibition scheme, arrows on the right side only indicates competitive inhibition, where vmax is unaffected.
• For an enzyme inhibition scheme, arrows on the left side only indicates uncompetitive inhibition, where both vmax and km decreases.
• For an enzyme inhibition scheme, arrows on the both sides indicates noncompetitive inhibition, where vmax decreases but km remains the same.
• Phosphatase removes phosphate, counteracted by kinase, which adds phosphate.
• These two enzymes often act in opposition to regulate cellular processes.
• Synthetases are ligases that link molecules together using ATP.
• Mutases are isomerases that rearrange groups within a molecule.
• To compare kcat (turnover), use kcat = Vmax / [E]
• If it's vitamin-derived and needed for the enzyme to work, it's a coenzyme.
• Transcription factors regulate gene expression by binding DNA and influencing transcription, they do not assist enzymes in catalyzing chemical reactions.
• Allosteric activators bind at sites other than the active site to increase enzyme activity.
• If a molecule binds at the active site, it is not allosteric.
• Oxidoreductases catalyze oxidation-reduction reactions of their substrates using cofactors.
• Cofactors can act as either reducing agents (e.g., NADH, FADH2, NADPH) or oxidizing agents (e.g., NAD+, FAD, NADP+).
• Transferases transfer functional groups from one molecule to another.
• Transferases receive two substrates and produce two products.
• Hydrolases catalyze hydrolysis reactions, which use water to break functional groups apart (e.g., phosphatase, gluconolactonase).
• Lyases break a single substrate into two products or join two substrates to make a single product.
• Lyases facilitate the breaking or formation of double bonds or a ring structure without using water, redox cofactors, or ATP.
• Isomerases rearrange covalent bonds within one molecule.
• Isomerase-catalyzed reactions can include functional group transfers (mutases), stereochemical rearrangement (racemases, epimerases), cis–trans rearrangement, and conformational rotation (chaperones).
• Ligases use the hydrolysis of a high-energy molecule (e.g., ATP) to join two substrates together.
• Ligases also have ATP hydrolase activity, but their primary activity is to join two substrates.
• Moderate heat increases enzyme activity by helping substrates reach the activation energy.
• Too much heat denatures enzymes, destroying their structure and function.
• If an enzyme works on several structurally related molecules, it is not highly specific.
• Reversible enzymes can often act on both product and substrate forms, especially when those products come in different stereoisomers.
• Western blots can report molecular weight and relative quantities of proteins but not their thermal stability.
• Heat capacity measurements, spectroscopy, and activity assays at different temperatures can measure thermal stability.
• Ligand binding to a transmembrane receptor enzyme (RTK) causes receptors to dimerize.
• Receptor dimerization leads to phosphorylation of intracellular domains and initiation of a downstream signaling cascade.
• Zymogens are inactive enzyme precursors activated by cleavage of part of their sequence, often unblocking the active site.
• Zymogens can autocleave or be cleaved by other enzymes.
• Apoenzymes are inactive enzymes without a cofactor.
• Holoenzymes are active enzyme + cofactor.
• Coenzymes are nonprotein molecules that help enzyme function (e.g., vitamins).
• In covalent catalysis, a nucleophilic amino acid side chain (like serine) attacks an electrophilic part of the substrate, forming a temporary covalent bond.
• Competitive inhibitors resemble the enzyme’s substrate and bind to the active site, often affecting multiple similar enzymes that share the same substrate recognition sequence.
• An inhibitor that resembles the substrate and reduces activity by binding the active site is a competitive inhibitor, not allosteric.
• Allosteric inhibitors bind elsewhere, not the active site, and change the enzyme's shape or function.
• Allosteric inhibitors don’t compete directly with the substrate.
• Irreversible inhibitors form covalent bonds with enzymes.
• Irreversible inhibitors become more effective with preincubation, allowing time for covalent linkages to form.
• Reversible inhibitors bind quickly and noncovalently.
• Preincubation does not increase the effect of reversible inhibitors.
• If preincubation increases inhibition, the inhibitor is likely irreversible (forms covalent bonds).
• If preincubation has no effect, the inhibitor is likely reversible (noncovalent interactions).
• Dephosphorylation can either activate or inactivate a protein, depending on the specific enzyme or signaling pathway involved.
• Competitive inhibitors bind E only.
• Uncompetitive inhibitors bind ES only.
• Mixed inhibitors bind both E and ES, though they may have a higher affinity for E than for ES, or vice versa.
• Noncompetitive inhibitors are a special case of mixed inhibitor in which the affinities for E and ES are exactly equal.
• Phosphorylation is a covalent modification that can either activate or inactivate an enzyme depending on the site and enzyme.
• If a reaction occurs after phosphorylation, that phosphorylation site likely activates the enzyme.
• If a reaction is blocked after phosphorylation, it likely inactivates the enzyme.