MODULE 6: Chapter 7.4 Flashcards
What is enzyme kinetics?
Enzyme kinetics is the quantitative study of the rates of chemical reactions performed by enzymes.
What is the primary goal of enzyme kinetics studies?
To develop an understanding of the characteristics of the enzyme with respect to a particular substrate.
What parameters can be determined from enzyme kinetics?
Kinetic parameters of the enzyme and substrate, effects of regulatory molecules, and effects of enzyme variants.
What does enzyme kinetics analyze?
Reaction rate data obtained with purified enzymes under defined laboratory conditions.
How does enzyme activity relate to temperature, pH, and substrate concentration?
Enzyme kinetics measures enzyme reaction rates quantitatively under varying conditions.
What is the formula for the velocity of a first-order reaction?
v = k[S]
What does the rate constant ‘k’ indicate in enzyme kinetics?
How quickly a substrate molecule is converted to product as a function of time.
What is the unit of the rate constant ‘k’ for a first-order reaction?
Second−1 (s−1)
What is the relationship between activation energy (ΔG‡) and reaction rate?
Lower ΔG‡ results in a higher reaction rate (v).
What characterizes a second-order reaction?
The reaction rate is proportional to the product of the substrate concentrations.
What are the units of a second-order rate constant?
Molarity−1 second−1 (M−1 s−1)
What is the Michaelis constant (Km)?
The substrate concentration at which the reaction rate is half of its maximum value (vmax).
What occurs at the maximum velocity (vmax) of an enzyme reaction?
The reaction velocity cannot increase further, even with additional substrate.
What is the significance of measuring initial velocity (v0) in enzyme kinetics?
It allows for the analysis of reaction rates before substrate concentration changes significantly.
What simplifying assumption is made in Michaelis–Menten kinetics regarding the enzyme-substrate complex?
The concentration of the enzyme-substrate (ES) complex remains relatively constant.
What is the equation used to describe Michaelis–Menten kinetics?
v0 = (vmax[S]) / (Km + [S])
True or False: Michaelis–Menten kinetics can be applied to enzyme reactions with multiple substrates.
True, but only one substrate can be varied at a time.
What is the role of the Boltzmann constant (kB) in enzyme kinetics?
It is used in the mathematical relationship between velocity and activation energy.
How is the concentration of the enzyme [E] related to the total enzyme concentration [Et]?
[Et] = [E] + [ES]
What does a low Km value indicate about an enzyme’s catalytic activity?
High catalytic activity at low substrate concentration.
What is the significance of the steady-state condition in enzyme kinetics?
The concentration of ES is constant while substrate decreases and product increases.
What experimental method is required to analyze the pre-steady-state condition?
Stop-flow kinetics.
What does the term ‘activation energy’ (ΔG‡) refer to?
The energy barrier that must be overcome for a reaction to occur.
Fill in the blank: Enzymes lower the _______ for reactions, increasing their rates.
activation energy (ΔG‡)
What is the equation relating enzyme concentration [E] and enzyme-substrate complex [ES]?
[E] = [Et] − [ES]
[Et] is the total enzyme concentration.
What does the equation k1[E][S] = (k₋1 + k2)[ES] represent?
The relationship between the enzyme concentration, substrate concentration, and the enzyme-substrate complex concentration.
What is the initial velocity of the reaction defined as?
v0 = k2[ES]
This equation indicates that the initial velocity depends on the concentration of the enzyme-substrate complex.
What does vmax represent in enzyme kinetics?
The maximum velocity of the reaction when all enzyme is in the form [ES].
What is the Michaelis–Menten equation?
It relates the initial velocity v0 to the substrate concentration, maximum velocity vmax, and the Michaelis constant Km.
What does Km indicate about an enzyme?
It is a value that characterizes the enzyme’s affinity for its substrate under specific conditions.
What is the significance of a high dissociation constant Kd?
It indicates that the enzyme has a low affinity for the substrate.
True or False: Km is a direct measure of enzyme affinity for substrate.
False
Km can be experimentally determined but does not always represent the dissociation constant Kd.
What is the Lineweaver–Burk equation used for?
To derive a double reciprocal plot of enzyme data for determining kinetic parameters.
What does the specificity constant kcat/Km represent?
It provides information about the catalytic efficiency of an enzyme.
Fill in the blank: The turnover number is defined as _______.
the number of conversions of substrate to product per unit time at a single active site when the enzyme is saturated.
What factors affect the rates of enzymatic reactions?
Both pH and temperature.
What is the optimal pH for pepsin?
Around 1.6.
How does temperature affect enzymatic reactions?
It influences both catalytic properties and protein structure stability.
What is the effect of high substrate concentration on v0 in Michaelis–Menten kinetics?
v0 does not increase appreciably at high substrate concentration.
What can enzyme kinetics reveal about enzyme mechanisms?
It provides insight into the role of the enzyme, its function in pathways, and potential therapeutic targets.
What is the relationship between Km and substrate concentration in enzyme kinetics?
Km is the substrate concentration at half-maximal velocity.
What is the primary limitation of the Michaelis–Menten model?
It is derived for single-substrate reactions and may not apply to multi-substrate reactions.
What is the significance of the diffusion-controlled limit in enzyme kinetics?
It represents the upper limit to the rate of reaction, approximately 10^8 to 10^9 M−1 s−1.
What does a high kcat value indicate about an enzyme?
That it has a high turnover rate.
Fill in the blank: The optimal temperature for a reaction balances _______ and structural stability.
catalytic efficiency
What is enzyme kinetics?
The quantitative study of the chemical reactions performed by enzymes.
Define a first-order reaction.
A reaction in which the rate varies as the first power of the reactant concentration.
What characterizes a second-order reaction?
A bimolecular reaction in which the rate is proportional to the product of the substrate concentrations.
What is the Michaelis–Menten kinetic model?
A model of reaction kinetics focusing on the formation of an enzyme–substrate complex and the subsequent catalytic step, at an early time when no appreciable product has been generated.
What is a steady-state condition in enzyme reactions?
A state of a reaction in which the concentration of product ES is relatively constant after an initial reaction time.
What occurs during the pre-steady-state condition?
An initial time in an enzyme reaction when enzyme–substrate complex formation is linear over time and changes in [S] are negligible.
What is stop-flow kinetics?
A very rapid enzyme assay method that permits measurements in the millisecond range.
What does the Michaelis–Menten equation describe?
The hyperbolic relationship between the initial reaction velocity v0 and the substrate concentration [S].
What is the Lineweaver–Burk equation?
An algebraic transformation of the Michaelis–Menten equation that allows enzyme data to be drawn as a double reciprocal plot.
What is the velocity of the reaction (v)?
The product of the rate constant of a reaction k and [S], the concentration of substrate.
Define the rate constant of a reaction (k).
A numerical constant that reflects how quickly a substrate molecule is converted to product as a function of time under a defined set of conditions.
What is initial velocity (v0)?
The reaction rate at the beginning of a reaction, before the substrate concentration has changed significantly.
What does maximum velocity (vmax) represent?
A point in a reaction where the reaction velocity cannot increase any further, even with the addition of more substrate.
What is the Michaelis constant (Km)?
A numerical value that relates the rate constants of breakdown and formation of the enzyme–substrate complex for a given enzyme reaction.
What is the turnover number (kcat)?
The maximum catalytic activity under saturating levels of substrate.
Define specificity constant (kcat/Km).
A ratio that measures the catalytic efficiency of two enzyme reactions or of the same enzyme with two different substrates.
What are the two primary mechanisms of enzyme regulation?
- Bioavailability regarding the amount of enzyme in different tissues and cellular compartments * Control of catalytic efficiency through protein modification.
What is the effect of positive regulatory mechanisms on enzyme activity?
They result in an overall increase in enzyme activity.
What is negative regulation in enzyme activity?
It results in a decrease in enzyme activity.
List the biochemical processes affecting enzyme bioavailability.
- RNA synthesis (gene transcription) * RNA processing * Protein synthesis * Protein degradation * Protein targeting.
What are the three primary mechanisms that affect catalytic efficiency?
- Binding of regulatory molecules * Covalent modification * Proteolytic processing.
What is enzyme inhibition?
A regulatory mechanism used to control enzyme activity.
What distinguishes reversible inhibition from irreversible inhibition?
- Reversible inhibition is due to noncovalent binding * Irreversible inhibition involves covalent bonding.
How can the effect of reversible inhibitors be decreased?
By diluting the enzyme reaction.
What is malonate’s role in enzyme inhibition?
It is a reversible inhibitor that competes with succinate for binding to succinate dehydrogenase.
Define suicide inhibitors.
A type of irreversible inhibitor that reacts with an enzyme during catalysis but fails to complete the reaction, remaining irreversibly bound.
What is a competitive inhibitor?
A molecule that inhibits substrate binding at the active site.
What happens to apparent Km in the presence of a competitive inhibitor?
The apparent Km value increases, reflecting the requirement of higher substrate concentration to reach vmax.
What do indinavir and saquinavir inhibit?
The HIV aspartate protease enzyme required for the maturation of viral proteins.
What do competitive inhibitors mimic in their structure?
Natural Phe-Pro dipeptide substrate
Where do competitive inhibitors bind?
Enzyme active site at the dimer interface
What type of inhibitors bind to enzyme–substrate complexes?
Uncompetitive inhibitors
What happens to the active site conformation due to uncompetitive inhibitors?
It becomes less catalytically active
Do uncompetitive inhibitors bind to the free enzyme?
No
What is the effect of uncompetitive inhibitors on Km and vmax?
Both decrease
What is the slope of the line Km/vmax in a Lineweaver–Burk plot affected by uncompetitive inhibition?
Unchanged
How do mixed inhibitors behave?
Bind to both the enzyme and the enzyme–substrate complex
What is a special case of mixed inhibition called?
Noncompetitive inhibition
What is the effect of noncompetitive inhibitors on Km?
Unchanged
What do allosteric regulators do?
Control enzyme activity through binding to sites other than the active site
What is feedback inhibition?
The end product of a pathway inhibits the first enzyme in that pathway
Which enzyme is a classic example of allosteric regulation?
Aspartate transcarbamoylase (ATCase)
What does ATCase catalyze?
Formation of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate
What is the role of CTP in relation to ATCase?
Feedback inhibitor
What activates ATCase?
ATP
What type of allosteric regulators are CTP and ATP?
CTP is negative, ATP is positive
What shape does the activity curve of cooperative enzymes like ATCase have?
Sigmoidal
What are homotropic allosteric activators?
Substrates that affect the binding at other active sites
What structural change occurs in ATCase upon binding of ATP?
Shifts to the activated R state
What is the effect of CTP binding to ATCase?
Stabilizes the inactive T-state conformation
What is the most common covalent modification in enzymes?
Phosphorylation of Ser, Thr, and Tyr residues
What do kinase enzymes do?
Add phosphoryl groups to enzymes
What removes phosphoryl groups from proteins?
Phosphatase enzymes
How does phosphorylation affect enzyme activity?
Can activate or inactivate enzymes
What is an example of an enzyme regulated by phosphorylation?
Glycogen phosphorylase
What is the molecular structure of glycogen phosphorylase in the phosphorylated R-state conformation?
It is a homodimer with pyridoxal phosphate bound in the active site and phosphorylated Ser residues at the subunit interface
The phosphorylated Ser residues are shown in yellow and are distinct from the active site
How does phosphorylation affect glycogen phosphorylase activity?
Phosphorylation shifts the enzyme from the inactive T-state conformation to the active R-state conformation
Phosphorylation of Ser14 by phosphorylase kinase activates the enzyme, while dephosphorylation by protein phosphatase 1 inactivates it
What is the role of phosphorylase kinase?
It catalyzes the phosphorylation of Ser14 in glycogen phosphorylase, stimulating its catalytic efficiency
Phosphorylase kinase is stimulated by the hormones glucagon and epinephrine
What does protein phosphatase 1 do in the context of glycogen phosphorylase?
It catalyzes the dephosphorylation of Ser14, shifting the equilibrium toward the inactive T-state
This enzyme is stimulated by the hormone insulin
What is adenylylation in the context of enzyme regulation?
A covalent modification involving the addition of AMP to tyrosine residues, regulating catalytic activity
An example is the inactivation of E. coli glutamine synthetase by adenylylation of Tyr397
How does uridylylation affect glutamine synthetase adenylyltransferase?
Uridylylation activates its deadenylylation activity, while deuridylylation activates its adenylation activity
This regulation is influenced by the levels of ATP, α-ketoglutarate, Pi, and glutamine in the cell
What is a zymogen?
An inactive enzyme precursor that requires proteolytic cleavage to become active
Examples include pepsinogen and chymotrypsinogen
What is the autocleavage reaction of pepsinogen?
It removes a 44-amino-acid N-terminal segment to generate the active enzyme pepsin
This reaction is stimulated by the acidic environment of the stomach
How is chymotrypsin activated from chymotrypsinogen?
Through four proteolytic cleavage events, starting with trypsin cleaving at Arg15
The final product is α-chymotrypsin, which consists of three polypeptide chains held by disulfide bonds
What is the mechanism of feedback inhibition?
The end product of a metabolic pathway inhibits the first enzyme in that pathway
This mechanism helps regulate metabolic processes efficiently
Define irreversible inhibition.
An enzyme regulatory mechanism in which an inhibitory molecule forms a covalent bond with catalytic groups in the enzyme active site
An example is diisopropylfluorophosphate, which inhibits protease and phospholipase enzymes
What characterizes competitive inhibition?
A molecule that inhibits substrate binding at the active site
This type of inhibition can be reversed by increasing substrate concentration
What is a suicide inhibitor?
An inhibitor that initiates the enzyme reaction mechanism but does not complete it, remaining covalently bound
This type of inhibitor effectively reduces enzyme activity
What is noncompetitive inhibition?
A special case of mixed inhibition where a molecule has equal affinity for both the free enzyme and the enzyme-substrate complex
This type of inhibition does not depend on substrate concentration
What is the function of kinases and phosphatases?
Kinases are phosphorylating enzymes, while phosphatases are dephosphorylating enzymes
They play crucial roles in the regulation of enzyme activity through reversible covalent modification
Fill in the blank: The three primary mechanisms of reversible regulation in the cell are _______.
competitive inhibition, allosteric regulation, covalent modification
