[1.1] Enzyme Kinetics and Regulation

Kinetics

General study of chemical reaction rates

Enzyme kinetics

The study of how the rates of enzyme-catalyzed chemical reactions are affected by changing the reaction conditions

• Involved factors like substrate concentration, pH, and temperature

Transition state

• Transient, high-energy intermediate

• Condition in which bonds in the substrate are maximally strained

• Highest energy point along the reaction pathway

  • Sits at the apex of the energy profile

  • Due to its high energy and instability, it has a fleeting existence and is the least probable configuration for molecules to adopt at any given time

• Can revert back to its reactants

Free energy change/Gibbs free energy (ΔG)

• Difference between the average free energy of the energies of the product and reactants/substrates for the given reaction

• Describes the direction the reaction will tend to proceed

• Only tells if a reaction will proceed or not

Negative ΔG value

• Reaction is favored from left to right

• Spontaneous

• Independent of the mechanism

Positive ΔG value

• Reaction is not favored

• Not spontaneous

• An energy input or coupling would be needed to drive it

Activation energy

• Energy input required to initiate the reaction

• Energy required to raise the average energy of 1 mol of reactant (at a given temperature) to transition-state energy

• The higher the activation energy, the slower the reaction

  • “Barrier” in transforming reactants to products

  • The energy that must be reached to form the products

True

True/False: Overall free energy change is not affected in the presence of enzymes; Gibbs free energy is independent of the mechanism.

False

True/False: The overall concentration of reactants and products does not remain constant. Hence, equilibrium constant is affected by enzymes.

Temperature

• Higher ________ → increasing kinetic energy → increased collision frequency of the reacting molecules

• Explained by the collision theory of capital kinetics

• “For two molecules to react, they must collide, and that they must have sufficient kinetic energy

Hydrogen ion concentration (pH)

• Affects active site ionization, enzyme denaturation, and rate of almost all enzyme-catalyzed reactions

• State of protonation is critical for substrate binding and catalysis

• Optimal levels differ for each enzyme

pH 5-9

pH at which most intracellular enzymes exhibit optimal activity

pH 2

pH at which pepsin (a gastric digestive enzyme) is maximally active

• If put in an alkaline environment (pH 9), it can be denatured

pH 7

pH at which chymotrypsin is maximally active

Substrate concentration

Increases with reaction rate until it reaches a maximum value (Vmax)

Vmax

• Point where all enzymes are saturated

• No further reaction can occur even as substrate concentration increases

• Rate already reached its maximum velocity

Rate is proportional to substrate concentration at low [S]

What happens to reaction rate at low substrate concentrations?

Rate becomes independent of substrate concentration at high [S]

What happens to reaction rate at high substrate concentrations?

Zero-order kinetics

What type of kinetics occurs when the enzyme is saturated with substrate?

Vmax

What maximal velocity does the reaction approach when the enzyme becomes saturated?

All enzyme active sites are occupied by substrate

What causes the saturation effect in zero-order kinetics?

Hyperbolic curve

What is the shape of the reaction velocity versus substrate concentration curve in Michaelis-Menten kinetics?

Michaelis constant (Km)

What does Km represent in Michaelis-Menten kinetics?

Substrate concentration at which vi = ½ Vmax

What is the definition of Km?

Low Km = high substrate affinity

What does a low Km indicate about enzyme affinity for its substrate?

High Km = low substrate affinity

What does a high Km indicate about enzyme affinity for its substrate?

[S] <<< Km

What substrate concentration condition produces first-order kinetics?

[S] >>> Km

What substrate concentration condition produces zero-order kinetics?

Lineweaver-Burk plot (double reciprocal plot)

What linear plot is used to analyze Michaelis-Menten kinetics?

It transforms the hyperbolic Michaelis-Menten curve into a straight line

What is the main purpose of the Lineweaver-Burk plot?

Km and Vmax

Which kinetic parameters can be determined from the Lineweaver-Burk plot?

Competitive inhibition: x-intercept unchanged, slope increased, y-intercept changed

How does competitive inhibition appear on a Lineweaver-Burk plot?

Enzymes with positive cooperativity

What type of enzymes is described by the Hill equation?

Binding of substrate to one site increases the affinity of the remaining sites

What is positive cooperativity?

Sigmoidal curve

What is the shape of the velocity versus substrate concentration curve for cooperative enzymes?

Multimeric enzymes with multiple substrate-binding sites

What type of enzymes can exhibit cooperative behavior?

Hill coefficient (nH)

What parameter indicates the degree of cooperativity in the Hill equation?

n = 1: binding sites behave independently

What does a Hill coefficient of 1 indicate?

n > 1: positive cooperativity

What does a Hill coefficient greater than 1 indicate?

Higher n = greater cooperativity and a more sigmoidal curve

What happens to the degree of cooperativity as the Hill coefficient increases?

Reversible inhibition involves non-covalent association or dissociation

What characterizes reversible enzyme inhibition?

Competitive inhibition

What type of reversible inhibition occurs when substrate and inhibitor compete for the same binding site?

Km increases; Vmax remains unchanged

What are the effects of competitive inhibition on Km and Vmax?

High substrate concentration

What can overcome the effects of competitive inhibition?

Statins, methotrexate, and malonate

What are examples of competitive enzyme inhibitors given in the lecture?

HMG-CoA reductase

What enzyme is competitively inhibited by statins?

Dihydrofolate reductase

What enzyme is competitively inhibited by methotrexate?

Succinate dehydrogenase

What enzyme is competitively inhibited by malonate?

Non-competitive inhibition

What type of reversible inhibition occurs when the inhibitor binds at a site distinct from the substrate-binding site?

Km remains unchanged; Vmax decreases

What are the effects of non-competitive inhibition on Km and Vmax?

No, increasing substrate concentration cannot overcome it

Can the effects of non-competitive inhibition be overcome by increasing substrate concentration?

Irreversible inhibition

What type of inhibition usually involves covalent modification of the enzyme?

Covalent bonds are formed with essential enzyme residues or prosthetic groups

How do irreversible inhibitors chemically modify enzymes?

Mechanism-based (suicide) inhibition

What type of irreversible inhibition involves a substrate analog that generates a reactive group during catalysis?

The reactive group forms a covalent bond with a functional group in the enzyme's active site

How does a mechanism-based inhibitor permanently inactivate an enzyme?

Penicillin

What irreversible inhibitor covalently reacts with an essential serine residue of glycopeptide transpeptidase?

Allopurinol

What drug inhibits xanthine oxidase through conversion to oxypurinol?

Oxypurinol binds the molybdenum cofactor of xanthine oxidase

How does allopurinol ultimately inactivate xanthine oxidase?

Bi-bi reaction

What is an enzymatic reaction involving two substrates and two reactions called?

Single-displacement reaction

What type of bi-bi reaction requires both substrates to combine with the enzyme before catalysis?

Ternary complex

What complex forms when both substrates bind to the enzyme in a single-displacement reaction?

Ordered mechanism

What single-displacement mechanism requires substrates to bind in a specific order?

Random mechanism

What single-displacement mechanism allows either substrate to bind first?

Double-displacement (ping-pong) mechanism

What bi-bi mechanism releases one or more products before all substrates have bound?

Transient modified form of the enzyme

What occurs to the enzyme during a double-displacement reaction?

Covalent catalysis

What type of catalysis is involved in the ping-pong mechanism?

To maintain homeostasis, avoid toxic metabolic products accumulation, and for energy conservation

Why is enzyme regulation important?

Cancer

What disease are overactive enzymes involved in?

Diabetes and Alzheimer’s

What other diseases are linked to enzyme problems?

Passive or active

What are the two main types of enzyme regulation?

Body responds to different substrate concentration or compartmentation

What characterizes passive regulation of enzymes?

Glycolytic pathway enzymes increase their activities

What happens to glycolytic pathway enzymes when glucose concentration is high after eating?

Glycolytic pathway enzymes are less active

What happens to glycolytic enzymes during starvation due to low glucose?

Pathway speeds up by increasing enzyme activity

How does the body respond to excessive nitrogenous waste in pathways like the urea cycle?

Certain metabolic pathways reside only within specialized metabolic cell types or subcellular compartments

What is compartmentation of enzymes?

Fatty acid synthesis occurs in the cytosol, while fatty acid oxidation occurs in the mitochondria

Where do fatty acid synthesis (anabolic) and fatty acid oxidation (catabolic) occur?

Places products and substrates in the mitochondria for easier access

Why is the TCA cycle compartmentalized in the mitochondria?

Cytochrome P-450 reductase and Cytochrome P450

What are the components of the microsomal ethanol oxidizing system?

Bottleneck or the slowest step in the pathway

What is a rate-limiting reaction in a metabolic pathway?

The enzyme catalyzing the rate-limiting reaction

What is a rate-limiting enzyme?

HMG-CoA reductase

Which rate-limiting enzyme do statin drugs inhibit in cholesterol synthesis?

Requires a certain signal to suddenly increase or decrease the quantity or activity of enzymes

What characterizes active regulation of enzymes?

Transcriptional regulation using inducers and repressors

How is enzyme synthesis controlled to change enzyme quantity?

Ubiquitin-proteasome pathway

What pathway controls the degradation of selected cellular proteins?

Regulate degradation of selected cellular proteins and remove defective or aberrant protein species

What is the function of protein degradation via the ubiquitin-proteasome pathway?

Defective proteins are tagged with ubiquitin molecules and sent to the proteasome for degradation

How does the ubiquitin-proteasome pathway degrade defective proteins?

Allosteric regulation and covalent modification

What are the two ways to increase or decrease the catalytic activity of an enzyme?

Activation or inhibition of enzyme activity through noncovalent interaction of the enzyme with effectors at an allosteric site

What is allosteric regulation?

Sigmoid-shaped curve

What type of curve do allosteric enzymes yield when reaction velocity is plotted against substrate concentration?

More than one polypeptide chain (subunit), each with a binding site for substrate and a distinct site for allosteric effectors

What is the structural characteristic of allosteric enzymes?

When an end product or intermediate inhibits or decreases the catalytic efficiency of an enzyme earlier in its pathway

What is feedback inhibition?

CTP

Which molecule acts as an allosteric feedback inhibitor of aspartate transcarbamoylase (ATCase) in pyrimidine biosynthesis?

ADP

Which molecule acts as an allosteric activator of ATCase to coordinate purine and pyrimidine pathways?

When an earlier metabolite activates an enzyme that catalyzes a reaction further down the pathway

What is feedforward activation?

Metabolic end products or intermediates with a secondary role as allosteric effectors extending beyond their pathway of origin

What are indicator metabolites?

Specialized allosteric ligands whose production or release is triggered in response to an external first messenger

What are second messengers?

Synthesized from ATP by adenylyl cyclase and activates Protein Kinase A (PKA)

How is cAMP produced and how does it function as a second messenger?

Introducing a new covalent bond or cleaving an existing one that alters the identity and properties of the affected enzyme

What is covalent modification of enzymes?

Partial proteolysis (transformation of proprotein into active forms)

What is an example of irreversible covalent modification?