Q:A: Enzymes: Properties, Kinetics, Inhibition, and Regulation

Flashcards covering enzyme kinetics, mechanisms, classification, inhibition, and regulation based on lecture slides.

What are enzymes defined as in terms of their molecular nature and functional effect on chemical reactions?

Enzymes are protein catalysts that increase reaction rates by lowering activation energy without altering the equilibrium of the reaction.

How does recommended enzyme nomenclature differ from systematic enzyme nomenclature?

Recommended names are short and informal, adding "-ase" to the substrate or reaction description, whereas systematic names are complete and formal, providing detailed information on function and substrate structure.

What is the key functional requirement that distinguishes a synthetase from a synthase?

A synthetase requires ATP, whereas a synthase does not require ATP.

How do oxidases and oxygenases differ regarding how oxygen is incorporated into products?

In oxidases, oxygen serves as an electron acceptor and no $O_2$ is incorporated into the product, whereas in oxygenases, $O_2$ is incorporated into the product.

What are the six major classes of enzymes according to the Enzyme Commission classification system?

1 Oxidoreductases, 2 Transferases, 3 Hydrolases, 4 Lyases ("synthases"), 5 Isomerases, and 6 Ligases ("synthetases").

What are the six important subclasses of Class 1 Oxidoreductases?

Dehydrogenases, Oxidases, Peroxidases, Reductases, Monooxygenases, and Dioxygenases.

What is an active site on an enzyme?

A special pocket formed by protein folding that contains amino acid residues that bind to the substrate and participate in catalysis.

What is an allosteric site on an enzyme?

A region different from the active site where regulators bind and cause conformational changes that alter the enzyme's reactivity.

By how much do enzymes typically speed up chemical reactions?

Enzymes speed up reaction rates by $10^3$ to $10^8$ times.

What four properties of the substrate binding site determine enzyme specificity?

Size, structure, charge, and hydrophobicity.

How do inorganic cofactors differ from coenzymes?

Cofactors are inorganic nonprotein components (such as $Fe^{2+}$ and $Zn^{2+}$), while coenzymes are organic nonprotein components derived from vitamins.

What are two examples of inorganic cofactors provided in the lecture?

$Fe^{2+}$ and $Zn^{2+}$.

What is a prosthetic group, and what specific example is cited in the notes?

A prosthetic group is a nonprotein moiety covalently linked to the enzyme, such as biotin.

What is the relationship between an apoenzyme, coenzyme/cofactor, and a holoenzyme?

An apoenzyme is an inactive enzyme lacking its nonprotein component, whereas a holoenzyme is the active enzyme complete with its nonprotein component ($\text{Apoenzyme} + \text{Coenzyme/Cofactor} = \text{Holoenzyme}$).

What is a zymogen or proenzyme, and what example is given in the lecture?

A zymogen (or proenzyme) is an inactive enzyme precursor, such as pepsinogen.

How is free activation energy defined and in what units is it measured?

Free activation energy is the energy difference between the substrate and the high-energy transition state formed prior to product formation, measured in $kcal/mole$.

How does lowering the activation energy ($E_a$) affect the reaction rate?

Lowering activation energy allows more substrate molecules to reach the transition state, causing the reaction to proceed quicker.

Do enzymes change the free energy levels of substrates or products?

No, enzymes lower activation energy without changing the free energy of substrates or products.

What occurs during substrate binding according to the induced-fit model?

Substrate binding to the active site causes conformational changes in the enzyme to form an enzyme-substrate complex.

How does stabilizing the transition state energy lead to increased reaction speed?

Stabilizing transition state energy at a lower level increases the concentration of intermediates that can be converted to products.

How is initial reaction velocity ($V_0$) defined and what are its units?

Initial reaction velocity ($V_0$) is the number of substrate molecules converted to product per unit of time, expressed in $mmol/min$.

Why does initial reaction velocity ($V_0$) plateau at high substrate concentrations?

Because all active sites on the enzyme become saturated with substrate at high substrate concentrations.

What happens to enzyme activity at temperatures exceeding $50\,^\circ\text{C}$?

Reaction velocity decreases rapidly due to temperature-induced enzyme denaturation.

What causes enzyme sensitivity to changes in pH?

pH sensitivity results from changes in the ionic charges of amino acid side chains required for optimal catalytic activity.

Based on their pH activity profiles, what are the optimal pH values for Pepsin, Lysozyme, and Alkaline Phosphatase?

Pepsin has an optimal pH around 2, Lysozyme around 5.5, and Alkaline Phosphatase around 10.

What are the two primary assumptions of the Michaelis-Menten kinetic model?

1) Substrate concentration is much greater than enzyme concentration ($[S] \gg [E]$). 2) Steady-state assumption: the rate of ES complex formation equals the rate of ES breakdown.

What is the Michaelis-Menten equation?

$$V_0 = \frac{V_{\max}[S]}{K_M + [S]}$$

What is the formula for the Michaelis constant ($K_M$) in terms of individual rate constants?

$$K_M = \frac{k_{-1} + k_2}{k_1}$$

How is $K_M$ defined in terms of substrate concentration?

$K_M$ is numerically equal to the substrate concentration ($[S]$) at which the reaction velocity reaches $\frac{1}{2}V_{\max}$.

What does a small $K_M$ indicate regarding an enzyme's affinity for its substrate?

A small $K_M$ reflects a high affinity of the enzyme for the substrate.

How is maximal velocity ($V_{\max}$) related to enzyme concentration ($[E]$)?

$V_{\max}$ is directly proportional to the concentration of enzyme ($[E]$).

What is a Lineweaver-Burk plot?

A double-reciprocal linear transformation of the Michaelis-Menten plot used to accurately calculate $V_{\max}$ and $K_M$.

What parameter is represented by the X-axis intercept of a Lineweaver-Burk plot?

$$-\frac{1}{K_M}$$

What parameter is represented by the Y-axis intercept of a Lineweaver-Burk plot?

$$\frac{1}{V_{\max}}$$

What parameter is represented by the slope of a Lineweaver-Burk plot?

$$\frac{K_M}{V_{\max}}$$

Identify the expressions for the slope, X-intercept, and Y-intercept shown on this Lineweaver-Burk plot.

Intercept on the X-axis = $-\frac{1}{K_M}$, Intercept on the Y-axis = $\frac{1}{V_{\max}}$, and Slope = $\frac{K_M}{V_{\max}}$.

How do reversible and irreversible inhibitors differ in their mode of chemical attachment to enzymes?

Irreversible inhibitors bind to enzymes through covalent bonds, whereas reversible inhibitors bind through non-covalent bonds.

What are the three types of reversible enzyme inhibition?

Competitive, Noncompetitive, and Uncompetitive inhibition.

Where do competitive inhibitors bind, and how do they interact with substrates?

Competitive inhibitors bind reversibly to the active site and compete directly with the substrate for binding.

How can competitive inhibition be overcome, and what is its effect on $V_{\max}$?

Competitive inhibition can be overcome by increasing substrate concentration ($[S]$); therefore, $V_{\max}$ remains unchanged.

What effect does a competitive inhibitor have on the apparent $K_M$?

A competitive inhibitor causes an apparent increase in $K_M$ for the substrate.

Where do the inhibited and uninhibited regression lines intersect on a Lineweaver-Burk plot during competitive inhibition?

They intersect on the Y-axis at $\frac{1}{V_{\max}}$.

What is the mechanism of action of statin drugs as competitive inhibitors?

Statins are structural analogs of the natural substrate for HMG-CoA reductase, competitively inhibiting cholesterol synthesis.

How does methotrexate act as a competitive inhibitor in medical therapy?

Methotrexate is a folic acid analog that inhibits dihydrofolate reductase, leading to reduced DNA synthesis.

How is ethanol used in treating methanol and ethylene glycol poisoning?

Ethanol acts as a competitive inhibitor by competing with methanol and ethylene glycol for binding to alcohol dehydrogenase.

Based on this structural diagram, why does pravastatin function as a competitive inhibitor of HMG-CoA reductase?

Pravastatin contains a chemical structure that mimics the active site-binding portion of the natural substrate HMG-CoA.

Where does a noncompetitive inhibitor bind relative to the active site?

A noncompetitive inhibitor binds to the free enzyme or the enzyme-substrate complex at a site different from the active site.

How does noncompetitive inhibition affect $V_{\max}$, and can this effect be reversed by adding substrate?

Noncompetitive inhibition causes an apparent decrease in $V_{\max}$ and cannot be overcome by increasing substrate concentration ($[S]$).

Why does $K_M$ remain unchanged in noncompetitive inhibition?

Because the noncompetitive inhibitor does not interfere with the affinity of the enzyme for its substrate.

Where do the inhibited and uninhibited regression lines intersect on a Lineweaver-Burk plot during noncompetitive inhibition?

They intersect on the X-axis at $-\frac{1}{K_M}$.

What is the therapeutic action of Angiotensin-converting enzyme (ACE) inhibitors?

They block plasma ACE to prevent cleavage of angiotensin I into angiotensin II (a potent vasoconstrictor), inducing vasodilation to lower blood pressure.

What enzyme does oxypurinol inhibit, and what metabolic pathway is blocked?

Oxypurinol (a metabolite of allopurinol) inhibits xanthine oxidase, blocking purine degradation to uric acid.

According to this summary table, compare competitive and noncompetitive inhibition in terms of $K_M$, $V_{\max}$, binding site, and substrate resemblance.

Competitive: $K_M$ increases, $V_{\max}$ is unchanged, binds active site ONLY, resembles substrate. Noncompetitive: $K_M$ is unchanged, $V_{\max}$ decreases, binds free enzyme OR E-S complex, does not resemble substrate.

What enzyme does aspirin irreversibly inhibit, and what product syntheses are blocked?

Aspirin irreversibly inhibits cyclooxygenase, blocking synthesis of thromboxane and prostaglandins.

What is the mechanism of action and physiological effect of Sarin nerve gas?

Sarin irreversibly inhibits acetylcholinesterase, blocking conversion of acetylcholine to choline and acetic acid, leading to continuous muscle stimulation.

What enzyme is irreversibly inhibited by the chemotherapeutic agent 5-Fluorouracil?

Thymidylate synthetase.

Provide an example of enzyme compartmentalization between the cytosol and mitochondria.

Enzymes responsible for fatty acid synthesis are located in the cytosol, whereas enzymes responsible for fatty acid oxidation are located in the mitochondria.

Based on this organelle compartmentalization diagram, which metabolic processes occur in the nucleus and lysosome?

Nucleus: DNA and RNA synthesis. Lysosome: Degradation of complex macromolecules.

What is the most common covalent modification for regulating enzymes, and which enzyme classes add and remove this group?

Phosphorylation; protein kinases add phosphate using ATP, and phosphoprotein phosphatases remove phosphate.

How does phosphorylation affect the activity of glycogen phosphorylase compared to glycogen synthase?

Glycogen phosphorylase is activated by phosphorylation, whereas glycogen synthase is inactivated by phosphorylation.

How do allosteric factors alter enzyme activity?

Allosteric factors bind non-covalently at a site different from the active site and induce conformational changes that alter substrate affinity ($K_M$) or catalytic activity ($V_{\max}$).

What is a homotropic effect in allosteric regulation?

A homotropic effect occurs when the substrate itself acts as a positive allosteric regulator, operating via cooperativity where binding at one site increases binding at other sites.

What is a heterotropic effect in allosteric regulation?

A heterotropic effect occurs when the allosteric effector is a molecule different from the substrate, often acting via feedback regulation where downstream products regulate upstream steps.

What are key characteristics of allosteric enzymes listed in the notes?

They catalyze rate-limiting reactions, have multiple subunits, do not follow Michaelis-Menten kinetics (show sigmoidal curves), and bind allosteric effectors.

What causes increased plasma levels of intracellular enzymes in clinical diagnosis?

Cell damage or cell necrosis resulting from disease or trauma, as well as increased cell proliferation.

Which specific plasma enzyme is measured as an indicator of liver damage?

Alanine aminotransferase (ALT).

What are isoenzymes (isozymes)?

Alternative enzyme forms composed of various subunit combinations that can be separated electrophoretically and catalyze similar reactions.

What two subunit types combine to form lactate dehydrogenase (LDH) isoenzymes?

H (heart) and M (muscle) subunits.

Based on this isoenzyme reference chart, state the subunit structures and primary tissue locations of $LDH_1$ and $LDH_5$.

$LDH_1$ ($H_4$) is highest in the heart and kidneys; $LDH_5$ ($M_4$) is highest in skeletal muscle and the liver.

What reaction equation and standard free energy change ($\Delta G'^\circ$) are associated with lactate dehydrogenase?

$\text{Pyruvate} + \text{NADH} + \text{H}^+ \rightleftharpoons \text{L-Lactate} + \text{NAD}^+$ with $\Delta G'^\circ = -25.1\,kJ/mol$.