6: Enzyme Kinetics, Allostery and Inhibition

Comprehensive practice flashcards covering enzyme kinetics, Michaelis-Menten dynamics, Lineweaver-Burk linearization, various types of inhibition, and regulatory mechanisms as presented in BMS 527 Lecture 6.

Who argued that biological processes are caused by chemical substances known as "ferments"?

Justus von Liebig

In what year was the name "enzyme" coined, and by whom?

It was coined in 1878 by Wilhelm Friedrich Kühne.

What does the Greek word "enzyme" literally translate to?

In yeast (en, in + zyme, yeast).

Who obtained a cell-free yeast extract capable of synthesizing ethanol from glucose in 1897?

Eduard Buchner

Most enzymes are predominantly composed of which macromolecule?

Proteins

Besides proteins, which other molecule is known to have catalytic activity?

RNA

What is the primary focus of the study of kinetics?

Kinetics is the study of the rates at which chemical reactions occur.

How does thermodynamics differ from kinetics in the context of enzymatic reactions?

Thermodynamics determines if a reaction is spontaneous (favorable), while kinetics indicates the reaction mechanism and path followed.

What four things can be determined through enzyme kinetic studies?

1. Binding affinities of substrates/inhibitors and maximum catalytic rate. 2. Catalytic mechanism. 3. The enzyme's role in a metabolic process. 4. The amount of enzyme present (assays).

Which scientist reported the rate of sucrose hydrolysis catalyzed by $\beta\text{-fructofuranosidase}$ in 1902?

Adrian Brown

In Adrian Brown's experiment, what parameters were kept constant and what was varied?

Enzyme concentration was kept constant while the substrate amount was varied.

What are the products of the hydrolysis of sucrose catalyzed by $\beta\text{-fructofuranosidase}$?

Glucose + Fructose

What happens to the reaction rate when the concentration of sucrose is much greater than the enzyme concentration?

The reaction rate becomes independent of the sucrose concentration (zero order).

In enzyme kinetics, what is the term for the amount of product formed over time?

Velocity ($v$)

In a "first order" reaction, the rate shows dependence on what?

The concentration of one component, typically the substrate.

What are the two elementary steps proposed by Adrian Brown for enzymatic reactions?

1. Substrate ($S$) forms a complex with enzyme ($E$) called the Enzyme-Substrate complex ($ES$). 2. The $ES$ decomposes into products ($P$) and $E$.

In the Michaelis-Menten model, when does the second step of the reaction become rate-limiting?

When the substrate concentration is high enough to entirely convert the enzyme to the $ES$ form.

What is the specific name of the Michaelis-Menten assumption stating that the concentration of $ES$ remains constant?

Steady-State

Mathematically, how is the "Steady-State" assumption for $ES$ formation and loss expressed?

$$K_1 + K_{-2} = K_{-1} + K_2$$

Which rate constant is typically excluded from the Michaelis-Menten description because it is considered very small?

$K_{-2}$ (the rate of $E + P \rightarrow ES$)

What is the assumption regarding product concentration ($[P]$) at early time points when measuring initial velocity ($V_o$)?

$$[P] \approx 0$$

What is the equation for the total enzyme concentration ($E_T$)?

$$[E_T] = [E] + [ES]$$

State the Michaelis-Menten Equation.

$$v = \frac{V_{max}[S]}{K_m + [S]}$$

What is the full name for the term represented by $K_m$?

Michaelis-Menten constant

By definition, what is the value of $[S]$ when $v = \frac{1}{2}V_{max}$?

$$K_m$$

What are the standard units for $V_{max}$?

$\text{concentration/time}$ (e.g., $\text{moles of product per minute}$)

What does a lower $K_m$ suggest about an enzyme's relationship with its substrate?

Higher affinity

What does a higher $K_m$ suggest about an enzyme's relationship with its substrate?

Lower affinity

Why is it "easy" to saturate an enzyme with a low $K_m$?

The enzyme reaches saturation at low substrate concentrations, so its activity doesn't vary much as substrate concentration changes.

What is the relationship between $[S]$ and velocity ($v$) when $[S] < K_m$?

Changes in $[S]$ greatly affect $v$, mimicking a first-order reaction.

What is the relationship between $[S]$ and velocity ($v$) when $[S] > K_m$?

Changes in $[S]$ have less effect on $v$, mimicking a zero-order reaction.

What is the standard unit of measurement for $K_m$?

$$\text{moles (concentration) of substrate}$$

What type of curve is produced when plotting Michaelis-Menten data as reaction rate against substrate concentration?

A hyperbolic curve

Which graphical method is used to linearize Michaelis-Menten data by plotting $1/v$ vs $1/[S]$?

Lineweaver-Burk plot

What does the y-intercept represent on a Lineweaver-Burk plot?

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

What does the gradient (slope) represent on a Lineweaver-Burk plot?

$$\frac{K_m}{V_{max}}$$

What does the x-intercept represent on a Lineweaver-Burk plot?

$$-\frac{1}{K_m}$$

If the x-intercept of a Lineweaver-Burk plot moves further to the left, what does it indicate about $K_m$?

The $K_m$ is smaller.

If the y-intercept of a Lineweaver-Burk plot moves upward, what does it indicate about $V_{max}$?

The $V_{max}$ is smaller.

What is the definition of $K_{cat}$ (turnover number)?

The measure of how many substrates a single enzyme converts to product per unit of time.

What is the mathematical formula for $K_{cat}$?

$$K_{cat} = \frac{V_{max}}{E_{total}}$$

How is catalytic efficiency calculated?

$$\text{Catalytic Efficiency} = \frac{K_{cat}}{K_m}$$

If Enzyme A and Enzyme B have the same $K_{cat}$ but Enzyme A has a lower $K_m$, which is more efficient?

Enzyme A

In mammals, enzyme velocity tends to double for every increase of how many degrees Celsius?

$10\,^{\circ}C$ (until reaching $37\,^{\circ}C$)

What happens to a protein's structure and enzyme activity after temperature exceeds the optimum range?

The protein denatures and activity falls off sharply.

Which enzyme in Siamese cats only functions below $36\,^{\circ}C$ to produce melanin in extremities?

Tyrosinase

How does pH affect enzymatic activity?

It affects the ionization states of amino acids and substrates, and extreme pH changes can denature proteins.

What is the optimum pH for the stomach enzyme pepsin?

$$pH\,2$$

What is the optimum pH for pancreatic enzymes?

$$pH\,8.5$$

What is the definition of an enzyme inhibitor?

A substance that reduces an enzyme's activity by combining with it to influence substrate binding or turnover.

What is the difference between a reversible and an irreversible inhibitor?

Reversible inhibitors are noncovalently bound and can be reversed, while irreversible inhibitors are covalently bound and cannot be reversed.

Define a competitive inhibitor.

A substance that competes directly with a normal substrate for an enzymatic binding site; it resembles the substrate but is unreactive.

How can competitive inhibition be completely reversed?

By adding substrate significantly in excess of the inhibitor ($[S] \gg [I]$).

Which drug is a competitive inhibitor of dihydrofolate reductase?

Methotrexate

Why is methotrexate used in cancer treatment?

It prevents the reduction of dihydrofolate to tetrahydrofolate (essential for DNA synthesis), killing rapidly dividing cancer cells.

What is the effect of a competitive inhibitor on $V_{max}$ and $K_m$?

$V_{max}$ is unchanged; $K_m$ is increased.

Where do the lines for an enzyme with and without a competitive inhibitor intersect on a Lineweaver-Burk (LWB) graph?

On the Y-axis ($1/V_{max}$).

Where does a non-competitive inhibitor bind on an enzyme?

An allosteric site (a site other than the active site).

What happens to the enzyme's active site when a non-competitive inhibitor binds to the allosteric site?

It causes a conformational change that prevents the conversion of substrate to product.

Why can substrate levels not mitigate the effect of a non-competitive inhibitor?

Because the inhibitor is not in direct competition with the substrate for the active site.

Cyanide acts as a non-competitive inhibitor for which specific enzyme?

Cytochrome C oxidase (Complex IV)

What is the effect of a non-competitive inhibitor on $V_{max}$ and $K_m$?

$V_{max}$ is decreased; $K_m$ is unchanged.

Where do the lines for an enzyme with and without a non-competitive inhibitor intersect on a Lineweaver-Burk (LWB) graph?

On the X-axis ($-1/K_m$).

Define an uncompetitive inhibitor.

An inhibitor that binds directly to the enzyme-substrate ($ES$) complex but not to the free enzyme ($E$).

What is the effect of an uncompetitive inhibitor on $V_{max}$ and $K_m$?

$V_{max}$ is decreased; $K_m$ is decreased.

Describe the appearance of the lines for an enzyme with and without an uncompetitive inhibitor on a Lineweaver-Burk graph.

The lines are parallel.

Define a mixed inhibitor.

An inhibitor that can bind to both the free enzyme ($E$) and the enzyme-substrate ($ES$) complex.

In mixed inhibition, what happens to $K_m$ if the inhibitor has a preference for the free enzyme?

$K_m$ increases (lower affinity for substrate).

In mixed inhibition, what happens to $K_m$ if the inhibitor has a preference for the $ES$ complex?

$K_m$ decreases (higher affinity for substrate).

Where do the lines for an enzyme with and without a mixed inhibitor intersect on a Lineweaver-Burk graph?

At a point not on either axis.

How does an irreversible inhibitor permanently inactivate an enzyme?

By bonding covalently to the active site.

What is Diisopropyl fluorophosphate (DIFP)?

A nerve gas that acts as an irreversible covalent inhibitor.

DIFP forms a complex with which specific amino acid residue at the active site of enzymes like acetylcholinesterase?

Serine (OH group)

Which enzyme responsible for stopping nerve transmission is inhibited by DIFP?

Acetylcholinesterase

How does Aspirin function as a covalent inhibitor?

It acts as an acetylating agent, covalently attaching an acetyl group to a serine residue in the active site of the $COX$ enzyme.

What percentage of known biochemical reactions involve two substrates and yield two products (bisubstrate reactions)?

$$\approx 60\%$$

What are the two most common types of bisubstrate reactions?

Transferase reactions and oxidation-reduction reactions.

In pharmacology, what is "Efficacy"?

The maximal effect a drug can produce ($V_{max}$ value); partial agonists have less efficacy than full agonists.

In pharmacology, what is "Potency"?

The amount of a drug needed for a given effect; it is unrelated to efficacy.

What is feed forward control in a metabolic pathway?

A metabolite produced early in a pathway speeds up the activity of an enzyme further down the pathway.

What is the difference between positive and negative feedback loops?

Positive feedback increases enzyme activity, while negative feedback decreases it.

What is phosphorylation, and which enzymes catalyze it?

The attachment of a phosphate group ($PO_4$) to a molecule, catalyzed by protein kinases.

Which amino acid residues can be phosphorylated in proteins?

Serine, threonine, or tyrosine (those with an OH functional group).

Which class of enzymes is responsible for removing phosphate groups?

Phosphatases

What is the effect of phosphorylation on enzyme activity?

It can either activate or inactivate the enzyme.