Enzymes and Catalysis: Biochemical Mechanisms and Classifications

What are enzymes?

Biologic polymers that are specialized proteins acting as molecular machines to make or break bonds.

How do enzymes affect chemical reactions?

They increase the rate of a chemical reaction without undergoing any permanent chemical change.

What is catalysis?

The process of increasing the rate of a chemical reaction, which can be enzymatic or chemical.

What are the benefits of enzymes in reactions?

Higher reaction rates, more specificity, and milder reaction conditions.

What role do enzymes play in biochemical processes?

They coordinate cellular metabolic needs and signaling networks.

How are enzymes used in diagnostics?

They serve as diagnostic tools in assays like Coupled Spectrophotometric assays, ELISA, and Immunohistochemistry.

What is the Enzyme Commission number?

A unique identifier given to each enzyme that indicates its classification and function.

What is the classification of enzymes based on their function?

Enzymes are classified into six main classes: Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and Ligases.

What do oxidoreductases do?

They transfer electrons, typically in the form of hydride ions or hydrogen atoms.

What is the function of transferases?

They transfer functional groups between donors and acceptors.

What do hydrolases do?

They transfer functional groups to water, often breaking down larger molecules.

What is the role of cofactors in enzyme activity?

Cofactors assist enzymes in catalysis, either by transiently associating or being tightly bound.

What is the transition state theory?

It describes the energy changes during a reaction, including the formation of transient unstable charges.

How do enzymes lower activation energy (ΔG‡)?

By following lower-energy reaction paths and forming transient covalent bonds and non-covalent interactions.

What is binding energy (ΔGB)?

The energy derived from enzyme-substrate interactions that stabilizes the activated complex and contributes to catalysis.

What is the Lock and Key Hypothesis?

It suggests that the specificity of an enzyme for its substrate arises from their geometrically complementary shapes.

What is the Induced Fit Hypothesis?

It proposes that the interaction between enzyme and substrate induces a conformational change in the enzyme, enhancing binding affinity.

What is reaction rate?

The speed at which a reaction occurs, influenced by factors like temperature and pH.

What is reaction equilibrium?

The state where there is no net conversion happening in the concentration of reactants or products.

What is the relationship between reaction rate and activation energy?

Reaction rate is inversely and exponentially related to activation energy (ΔG‡).

What are ligases?

Enzymes that catalyze synthetic reactions where two molecules are joined, often using ATP.

What do peroxidases do?

They utilize hydrogen peroxide (H2O2) as a substrate in their reactions.

What is the role of metal ions in enzyme activity?

Metal ions can act as cofactors, either activating enzymes or being tightly bound as metalloenzymes.

What is substrate specificity?

The ability of an enzyme to preferentially bind to a specific substrate over others.

What is the significance of weak interactions in enzyme catalysis?

Weak interactions between enzyme and substrate are optimized in the transition state, driving the reaction forward.

What is the effect of enzyme concentration on reaction rate?

Increasing enzyme concentration typically increases reaction rate, assuming substrate is in excess.

What are the major moieties transferred by transferases?

Amino, phospho, acyl, one-carbon, and glycosyl groups.

What is the role of the active site in enzymes?

The active site binds substrates and facilitates catalysis through specific interactions.

How does enzyme specificity relate to reaction outcomes?

Enzyme specificity ensures that only certain substrates are converted into products, influencing metabolic pathways.

What is the role of enzymes in chemical reactions?

Enzymes increase the reaction rate without affecting the reaction equilibrium.

What does the equilibrium constant K'eq represent in a reaction?

K'eq = [P]/[S], where P is the product and S is the substrate.

How does ΔG'o relate to reaction equilibrium?

More negative ΔG'o implies a more favorable reaction equilibrium.

What factors affect enzyme-catalyzed reactions?

Substrate concentration, product concentration, enzyme concentration, enzyme activity, pH, and temperature.

What is the significance of collision theory in reactions?

For a reaction to occur, molecules must collide with sufficient kinetic energy and be within bond-forming distance.

How does temperature affect enzyme activity?

Raising temperature increases kinetic energy and collision frequency, thus increasing reaction rate, but extreme temperatures can denature enzymes.

What is the Q10 in enzyme kinetics?

Q10 is the factor by which the rate of a biological process increases for a 10 °C increase in temperature.

What pH range do most enzymes operate effectively within?

Typically between pH 5 to 9.

How does pH affect enzyme activity?

Changes in pH affect substrate ionization, enzyme binding, and the ionization of amino acids in the active site.

What is Km (Michaelis Constant)?

Km is the substrate concentration at which the reaction velocity (V0) is half of Vmax.

What does a low Km indicate about an enzyme's efficiency?

A low Km means the enzyme achieves maximal catalytic efficiency at low substrate concentrations.

What is the Michaelis-Menten assumption regarding enzyme kinetics?

It assumes that the formation of the enzyme-substrate complex (ES) reaches equilibrium quickly compared to the breakdown to product.

What is the significance of Vmax in enzyme kinetics?

Vmax is the maximum velocity of an enzyme-catalyzed reaction when the enzyme is saturated with substrate.

What does the Lineweaver-Burk plot help determine?

It helps assess Vmax and Km by using the reciprocal of reaction velocity and substrate concentration.

What is the turnover number (kcat) of an enzyme?

The maximal number of substrate molecules converted to product by one enzyme molecule per second.

What is the specificity constant (kcat/Km)?

It measures an enzyme's catalytic efficiency when substrate concentration is much lower than Km.

What happens to enzyme activity at temperatures above 45-55 °C?

Enzymes may begin to denature, leading to a loss of catalytic activity.

How does substrate concentration affect reaction velocity at low [S]?

At low substrate concentrations, velocity (V0) increases linearly with an increase in [S].

What occurs when substrate concentration is very high?

The reaction velocity approaches Vmax and becomes insensitive to further increases in substrate concentration.

What does the term 'steady state' refer to in enzyme kinetics?

It refers to the condition where the concentration of the enzyme-substrate complex (ES) remains constant during the reaction.

What is the effect of temperature on enzyme stability?

Enzymes from thermophilic microorganisms can remain stable at temperatures above 100 °C.

What is the relationship between kcat and enzyme activity?

kcat is the rate constant for the conversion of ES to product, reflecting the enzyme's catalytic activity.

What does a high specificity constant indicate?

It indicates that the enzyme is highly efficient at converting substrate to product.

How does enzyme concentration affect reaction rate?

Increasing enzyme concentration generally increases the reaction rate, provided substrate is not limiting.

What is the impact of reversible reactions on enzyme kinetics?

Reversible reactions can complicate the measurement of reaction rates and enzyme activity.

How does the presence of product (P) affect enzyme kinetics?

Product accumulation can inhibit enzyme activity, affecting the overall reaction rate.

What is the significance of the pKa shift in amino acids within enzymes?

The pKa of amino acid side chains can be altered in the protein environment, affecting enzyme activity.

What is the role of the gas constant (R) in enzyme kinetics?

R is used in calculations involving temperature and energy changes in reactions.

What is a first-order reaction?

A reaction where the rate is proportional to the concentration of one reactant (S), expressed as v = k[S].

What characterizes a second-order reaction?

A reaction where the rate is proportional to the square of the concentration of one reactant (S + S → P) or the product of the concentrations of two reactants (S1 + S2 → P).

What are Bi-Bi reactions?

Reactions that involve two substrates and typically occur in sequential or ping-pong mechanisms.

Define Maximum Velocity (Vmax).

The rate of reaction under optimal conditions when the enzyme is saturated with substrate.

What is the Michaelis Constant (Km)?

The substrate concentration at which the reaction velocity is half of Vmax.

What does the catalytic constant (Kcat) represent?

The number of substrate molecules converted to product per enzyme molecule per unit time.

How is enzyme activity measured?

By the amount of substrate converted to product per minute, expressed in international units (IU) or μmol/min.

What is specific activity?

The activity of an enzyme per milligram of total protein, expressed as U/mg protein.

What is a nucleophile?

A chemical species that donates a pair of electrons to an electrophile.

What is an electrophile?

An atom or molecule that is electron deficient and seeks an electron pair to form a covalent bond.

What is competitive inhibition?

Inhibition where a substance competes with the substrate for the active site of the enzyme.

How can competitive inhibition be overcome?

By increasing the concentration of the substrate.

What is uncompetitive inhibition?

Inhibition where the inhibitor binds to the enzyme-substrate complex, preventing the reaction from proceeding.

What is mixed inhibition?

Inhibition where the inhibitor can bind to both the enzyme and the enzyme-substrate complex, affecting both Km and Vmax.

What is irreversible inhibition?

Inhibition where the inhibitor covalently binds to or destroys an essential functional group on the enzyme.

What is a zymogen?

An inactive precursor form of an enzyme that requires cleavage to become active.

What is allosterism?

A non-covalent change in enzyme activity due to the binding of an effector molecule at a site other than the substrate-binding site.

What role does the enzyme alcohol dehydrogenase play in methanol poisoning?

It has a higher affinity for ethanol, which can outcompete methanol and reduce toxic metabolite formation.

What is the principle of enzyme-linked immunosorbent assays (ELISA)?

A technique used to detect proteins lacking catalytic activity using antibodies linked to enzymes.

What are spectrophotometric assays?

Assays that measure the ability of a substrate or product to absorb light, often used to estimate enzyme activity.

What is the significance of Km in enzyme kinetics?

It indicates the substrate concentration required for the enzyme to reach half of its maximum velocity.

What is the effect of irreversible inhibitors on enzymes?

They permanently inactivate enzymes by forming stable covalent bonds or strong non-covalent associations.

What is the relationship between enzyme concentration and reaction velocity?

As enzyme concentration increases, the reaction velocity increases until substrate saturation is reached.

What are the non-covalent forces involved in enzyme-substrate interactions?

Van der Waals forces, electrostatic interactions, hydrogen bonding, and hydrophobic interactions.

What happens at the maximum velocity of an enzyme-catalyzed reaction?

All enzyme molecules are in the enzyme-substrate complex form, and the concentration of free enzyme is zero.

What is the significance of enzyme activity in biochemical research?

It helps in understanding enzyme function, regulation, and potential therapeutic targets.

What are positive and negative effectors in allosterism?

Positive effectors are allosteric activators that enhance activity, while negative effectors are allosteric inhibitors that reduce activity.

What is homotropic allosteric regulation?

Regulation where the active site and allosteric sites are the same, leading to cooperative binding.

What is heterotropic allosteric regulation?

Regulation where the active site and allosteric sites are different, affecting enzyme activity through different molecules.

What is feedback inhibition?

A regulatory mechanism where the end product of a metabolic pathway inhibits an earlier step, coordinating cellular metabolic needs.

What is the significance of isoleucine in feedback inhibition?

Isoleucine specifically inhibits threonine dehydratase, demonstrating heterotropic allosteric inhibition.

How does allosteric enzyme kinetics differ from Michaelis-Menten kinetics?

Allosteric enzymes exhibit sigmoid kinetic behavior due to cooperative interactions, unlike the hyperbolic behavior of Michaelis-Menten kinetics.

What is the role of aspartate transcarbamoylase (ATCase) in pyrimidine nucleotide biosynthesis?

ATCase catalyzes the synthesis of N-carbamoyl aspartate from carbamoyl phosphate and aspartate, exhibiting cooperative binding and allosteric regulation.

What is the effect of ATP and CTP on ATCase?

ATP acts as a heterotropic activator, while CTP acts as a heterotropic inhibitor, coordinating the synthesis of purine and pyrimidine nucleotides.

What are the six catalytic mechanisms leading to rate enhancement?

1. Proximity and orientation effects 2. Catalysis by strain 3. Electrostatic catalysis 4. Metal ion catalysis 5. Acid-base catalysis 6. Covalent catalysis.

How does proximity and orientation effect enhance reaction rates?

By increasing the local concentration of substrates and spatially orienting them in an ideal position for interaction.

What is catalysis by strain?

Binding substrates in a conformation that creates strain, making bonds more vulnerable to cleavage.

What is electrostatic catalysis?

Exclusion of water from the active site enhances electrostatic interactions, stabilizing transition states and guiding substrates.

What is acid-base catalysis?

A mechanism where certain amino acid side chains act as general acids or bases to facilitate biochemical reactions.

How does HIV protease utilize acid-base catalysis?

Aspartic acid residues act as a general base and acid to activate water and facilitate peptide bond hydrolysis.

What is metal ion catalysis?

Involves metal ions stabilizing transition states and facilitating reactions, such as in the case of enolase with Mg2+.

What is covalent catalysis?

A mechanism where the enzyme forms a transient covalent bond with the substrate, often involving nucleophilic attack.

What is the role of chymotrypsin in peptide bond cleavage?

Chymotrypsin is a serine protease that uses covalent catalysis and a catalytic triad to cleave peptide bonds.