Biochemistry I - CHM219: Enzyme Kinetics

Induced fit describes the enzyme changing shape upon substrate binding, while conformational selection suggests the enzyme already exists in multiple conformations, and the substrate binds to a specific pre-existing conformation.

The initial rate of an enzyme-catalyzed reaction is calculated under the assumption that the reverse reaction is negligible and the rate-determining step is the conversion of substrate to product. This simplifies the rate equation.

kcat represents the apparent rate constant for the rate-determining step of converting substrate to product in an enzyme-catalyzed reaction.

The reaction velocity is defined as the observed rate of product formation, and it's directly proportional to the concentration of the enzyme-substrate complex [ES] and the kcat value. The reaction is assumed to be a simple first-order reaction.

The concentration of the enzyme-substrate complex [ES] is difficult to measure directly. Therefore, it is desirable to express the reaction rate in terms of the substrate concentration [S] and the total enzyme concentration.

The steady state assumption assumes that the concentration of the enzyme-substrate complex [ES] remains relatively constant over time even though the reaction is proceeding. This assumption is valid under certain conditions.

The equilibrium dissociation constant Kd describes the equilibrium between the free enzyme (E), the free substrate (S), and the enzyme-substrate complex (ES). It reflects the affinity of the enzyme for the substrate.

The steady state assumption simplifies the analysis of enzyme kinetics by considering the concentration of the enzyme-substrate complex [ES] to be constant over time. It is a useful approximation, but not always accurate.

A graphical method for analyzing enzyme kinetics. Plotting reaction velocity (v) against the ratio of velocity to substrate concentration (V/[S]) provides a linear representation of enzyme activity.

A graphical method for analyzing enzyme kinetics. Plotting the reciprocal of reaction velocity (1/v) against the reciprocal of substrate concentration (1/[S]) provides a linear representation of enzyme activity.

A change in the rate constant (kcat) resulting from an amino acid mutation in the enzyme active site.

A change in the Michaelis constant (KM) resulting from an amino acid mutation in the enzyme active site.

A type of enzyme reaction mechanism where the enzyme binds substrates in a specific order. One substrate must bind first before the other can bind.

The maximum rate at which an enzyme can catalyze a reaction.

A type of enzyme inhibition where the inhibitor binds to the active site of the enzyme, competing with the substrate.

A type of enzyme reaction mechanism where the binding of one substrate can affect the binding of other substrates. The order of substrate binding is not fixed.

A type of enzyme inhibition where the inhibitor binds to a site on the enzyme different from the active site, affecting the enzyme's ability to bind substrate and/or catalyze the reaction.

A type of enzyme inhibition where the inhibitor binds to both the free enzyme and the enzyme-substrate complex, reducing the enzyme's activity in both cases.

A type of enzyme inhibition where the inhibitor binds irreversibly to the enzyme, usually by forming a covalent bond.

A small molecule that is required for an enzyme to function properly.

A type of cofactor that is derived from vitamins.

A type of cofactor that is a metal ion, such as magnesium or zinc.

A coenzyme that carries electrons and hydrogen ions (H+) in metabolic reactions. It can be reduced to NADH by accepting two electrons and a proton, acting as an oxidizing agent. The reaction is also reversible, allowing NADH to act as a reducing agent.

Enzymes that catalyze oxidation-reduction reactions, where electrons are transferred.

Enzymes that transfer functional groups from one molecule to another.

Enzymes that break down molecules by adding water (hydrolysis).

Enzymes that remove a group from or add a group to a double bond, or other cleavages involving electron rearrangement.

Enzymes that catalyze intramolecular rearrangement, changing the structure of a molecule.

Enzymes that join two molecules together.

A system used to classify enzymes, assigning a four-part number to each enzyme based on its function. The first three numbers represent major class, subclass, and sub-subclass respectively.

A process where a molecule becomes catalytically active after being cleaved by an enzyme.

A series of protease activations leading to the conversion of fibrinogen to fibrin, forming a blood clot.

The process of removing fibrinopeptides A and B from fibrinogen by thrombin, exposing sites for fibrin monomers to associate and form a fiber.

A reversible covalent modification where a kinase adds a phosphate group to a protein, and a phosphatase removes it.

An example of irreversible enzyme activation by proteolytic cleavage. A series of cleavages in chymotrypsinogen lead to the active form of chymotrypsin.

The rotation of regulatory subunits in an enzyme, causing the catalytic subunits to move apart and rotate, leading to a change in activity.

A covalent modification where a protein is cleaved by an enzyme, permanently altering its structure and function.

A specific type of proteolytic cleavage where a precursor molecule is activated by removing a portion of its structure.

The rate of a reaction is affected by the concentration of substrate, and enzymes can show cooperative binding, meaning the binding of one substrate molecule can influence the binding of others. In cooperative binding, the enzyme doesn't follow Michaelis-Menten kinetics, and the graph of reaction velocity against substrate concentration is sigmoidal (S-shaped).

Allosteric enzymes are regulated by molecules that bind to a site different from the active site, called the regulatory site. These molecules, known as effectors, can be activators or inhibitors, influencing the enzyme's activity. Activators shift the equilibrium towards the R state, while inhibitors stabilize the T state, thus altering the enzyme's affinity for the substrate.

The T state is a low-affinity state with a higher Km, while the R state represents a high-affinity state with a lower Km. This means the T state requires a higher substrate concentration to reach half the maximal velocity compared to the R state.

In the absence of any activators or inhibitors, the allosteric enzyme shows sigmoidal kinetics due to the cooperative binding of the substrate. The sigmoidal curve indicates a switch-like behavior, transitioning from low activity to high activity over a narrow range of substrate concentrations.

In the synthesis of pyrimidine nucleotides, the enzyme aspartate carbamoyltransferase (ATCase) plays a crucial role by catalyzing the first committed step of the pathway. This reaction involves the combination of carbamoyl phosphate and aspartate to form N-carbamoyl-L-aspartate. The activity of ATCase is strictly regulated by the cell to ensure proper control of pyrimidine nucleotide production.

In prokaryotes, the aspartate carbamoyltransferase (ATCase) is a key regulatory point for pyrimidine biosynthesis. However, in most eukaryotes, the regulation of pyrimidine synthesis occurs at a preceding step, catalyzed by carbamoyl phosphate synthetase II.

ATP acts as an activator of ATCase, enhancing the enzyme's activity and promoting pyrimidine synthesis. CTP, on the other hand, acts as an inhibitor of ATCase, slowing down the reaction and preventing overproduction of pyrimidine nucleotides.

ATCase is an allosteric enzyme that exists in two conformations: the T state (inactive) and the R state (active). The T state has low affinity for substrate, while the R state has high affinity. The shift between these states is regulated by allosteric effectors like ATP and CTP.