Enzyme Kinetics

General Properties of Enzymes

Enzymes are biological catalysts that accelerate chemical reactions in living organisms. They are highly specific for their substrates and operate under mild physiological conditions.

• Specificity: Enzymes typically catalyze only one type of reaction or act on a specific substrate.

• Efficiency: Enzymes can increase reaction rates by factors of 106 or more.

• Regulation: Enzyme activity can be regulated by various mechanisms, including allosteric control and covalent modification.

Single Substrate Enzyme Kinetics

Single substrate enzymes follow characteristic kinetic behavior, often described by the Michaelis-Menten model.

• Substrate Binding: The enzyme binds to its substrate to form an enzyme-substrate (ES) complex.

• Product Formation: The ES complex is converted to product, releasing the enzyme for another catalytic cycle.

Transition State Stabilization

Enzymes lower the activation energy of reactions by stabilizing the transition state, the high-energy intermediate between reactants and products.

• Transition State: The configuration of atoms at the maximum energy point along the reaction coordinate.

• Stabilization: Enzymes provide an environment that favors the formation of the transition state, often through specific interactions with substrate functional groups.

Michaelis-Menten Equation

The Michaelis-Menten equation describes the rate of enzymatic reactions as a function of substrate concentration:

• Equation:

• v: Initial reaction velocity

• Vmax: Maximum velocity

• [S]: Substrate concentration

• Km: Michaelis constant, substrate concentration at half-maximal velocity

Plot: The Michaelis-Menten plot is hyperbolic, showing velocity versus substrate concentration.

Pre-Steady-State and Steady-State Kinetics

• Pre-Steady-State: The initial phase of the reaction before the ES complex concentration becomes constant.

• Steady-State: The phase where the formation and breakdown of ES complex are balanced, and the reaction rate is constant.

Enzymatic Kinetic Parameters

• Km (Michaelis Constant): Indicates the affinity of the enzyme for its substrate; lower Km means higher affinity.

• kcat (Turnover Number): Number of substrate molecules converted to product per enzyme molecule per unit time.

• Vmax (Maximal Velocity): The maximum rate achieved by the system at saturating substrate concentration.

• kcat/Km: A measure of catalytic efficiency.

Michaelis-Menten and Lineweaver-Burk Plots

• Michaelis-Menten Plot: Plots v versus [S]; used to estimate Vmax and Km.

• Lineweaver-Burk Plot: Double reciprocal plot ( vs ) linearizes the data:

• Intercepts and slopes allow determination of kinetic parameters.

Allosteric Activators and Inhibitors

• Allosteric Activator: A molecule that increases enzyme activity by binding to a site other than the active site.

• Allosteric Inhibitor: A molecule that decreases enzyme activity by binding to an allosteric site.

• Allosteric enzymes often display sigmoidal (S-shaped) kinetics rather than hyperbolic.

Enzyme Mechanisms

Exergonic vs. Endergonic Reactions

• Exergonic: Reactions that release free energy (); spontaneous.

• Endergonic: Reactions that require input of energy (); non-spontaneous.

• Reaction coordinate diagrams illustrate energy changes during reactions.

Proximity Effect

• Enzymes increase reaction rates by bringing substrates into close proximity and correct orientation, facilitating bond formation or cleavage.

Lock-and-Key vs. Induced-Fit Models

• Lock-and-Key: The enzyme active site is complementary in shape to the substrate.

• Induced-Fit: The enzyme changes shape upon substrate binding to better fit the substrate.

Transition State Stabilization

• Enzymes stabilize the transition state through specific interactions, lowering activation energy.

Serine Protease Catalysis and Specificity

• Catalytic Triad: A set of three amino acids (Ser, His, Asp) in the active site that work together to hydrolyze peptide bonds.

• Substrate Binding: Specificity is determined by the shape and chemical properties of the binding pocket.

Chymotrypsin Mechanism

• Chymotrypsin is a serine protease that cleaves peptide bonds adjacent to aromatic amino acids.

• Mechanism involves formation of a covalent acyl-enzyme intermediate and stabilization of the transition state by the oxyanion hole.

Enzyme Inhibition

Types of Inhibition

• Competitive Inhibition: Inhibitor binds to the active site, competing with the substrate. Increases Km, Vmax unchanged.

• Uncompetitive Inhibition: Inhibitor binds only to the ES complex. Decreases both Km and Vmax.

• Noncompetitive Inhibition: Inhibitor binds to both the enzyme and ES complex at a site other than the active site. Vmax decreases, Km unchanged.

• Lineweaver-Burk plots can distinguish inhibition types by their characteristic changes in slope and intercepts.

Mechanism-Based Inhibitors

• Also called "suicide inhibitors," these bind irreversibly to the enzyme, often by mimicking the transition state or forming a covalent bond during catalysis.

Cofactors

Essential Ions vs. Coenzymes

• Essential Ions: Metal ions required for enzyme activity (e.g., Mg2+, Zn2+).

• Coenzymes: Organic molecules that assist enzymes; can be tightly (prosthetic groups) or loosely (cosubstrates) bound.

Types of Coenzymes

• Cosubstrates: Transiently associated with the enzyme (e.g., NAD+).

• Prosthetic Groups: Permanently attached to the enzyme (e.g., FAD).

ATP in Group Transfer Reactions

• ATP acts as a cofactor by donating phosphate groups in phosphorylation reactions.

NAD(P)+/NAD(P)H and FAD/FADH2 in Redox Reactions

• NAD+ and FAD are electron carriers, accepting electrons during oxidation reactions and donating them during reduction.

Monosaccharides

Functions of Carbohydrates

• Energy storage (e.g., glucose, glycogen)

• Structural components (e.g., cellulose, chitin)

• Cell recognition and signaling (e.g., glycoproteins)

Definitions

• Monosaccharides: Simple sugars (e.g., glucose, fructose)

• Disaccharides: Two monosaccharides linked by a glycosidic bond (e.g., sucrose)

• Oligosaccharides: 3–10 monosaccharide units

• Polysaccharides: Long chains of monosaccharides (e.g., starch, cellulose)

Aldoses vs. Ketoses

• Aldoses: Monosaccharides with an aldehyde group (e.g., glucose)

• Ketoses: Monosaccharides with a ketone group (e.g., fructose)

Fischer Projections and Chirality

• Fischer projections are two-dimensional representations of sugars, showing the configuration of chiral centers.

• Chirality is determined by the arrangement of groups around asymmetric carbons.

Stereochemistry of Monosaccharides

• Stereoisomer: Same molecular formula, different spatial arrangement.

• Enantiomer: Non-superimposable mirror images.

• Diastereomer: Stereoisomers that are not mirror images.

• Epimer: Differ at only one chiral center.

Cyclization of Monosaccharides

• Monosaccharides can cyclize to form hemiacetals (aldoses) or hemiketals (ketoses).

• The new chiral center formed is called the anomeric carbon.

• Haworth projections depict the cyclic forms of sugars.

• Pyranose: Six-membered ring; Furanose: Five-membered ring.

Disaccharides

Glycosidic Bonds and Disaccharide Formation

• Disaccharides are formed by condensation reactions between the anomeric carbon of one monosaccharide and a hydroxyl group of another, creating a glycosidic bond.

• Reducing sugars: Have a free anomeric carbon; Non-reducing sugars: Both anomeric carbons are involved in the glycosidic bond.

• Common disaccharides: Sucrose (glucose + fructose), Lactose (glucose + galactose).

Polysaccharides and Glycoproteins

Polysaccharide Structure and Function

• Starch: Storage polysaccharide in plants; mixture of amylose (linear) and amylopectin (branched).

• Glycogen: Storage polysaccharide in animals; highly branched.

• Cellulose: Structural polysaccharide in plants; linear chains of glucose with β(1→4) linkages.

Glycoproteins

• Proteins with covalently attached carbohydrate chains.

• O-linked: Carbohydrate attached to the hydroxyl group of serine or threonine.

• N-linked: Carbohydrate attached to the amide nitrogen of asparagine.

• Glycoproteins play roles in cell recognition, signaling, and immune response.

ABO Blood Classification

• Blood types are determined by specific oligosaccharide structures on red blood cell surfaces.

• Compatibility between donor and recipient depends on the presence or absence of A and B antigens.

Nucleic Acids

Components of a Nucleotide

• Nucleobase: Purine (adenine, guanine) or pyrimidine (cytosine, thymine, uracil).

• Sugar: Ribose (RNA) or deoxyribose (DNA).

• Phosphate group: Attached to the 5' carbon of the sugar.

Nucleobases, Nucleosides, and Nucleotides

• Nucleobase: Nitrogenous base only.

• Nucleoside: Base + sugar.

• Nucleotide: Base + sugar + phosphate.

Structures and Codes

• Purines: Adenine (A), Guanine (G)

• Pyrimidines: Cytosine (C), Thymine (T, DNA only), Uracil (U, RNA only)

• One-letter codes: A, G, C, T, U

• Phosphoanhydride bonds: Between phosphate groups (e.g., in ATP)

• Phosphoester bonds: Between phosphate and sugar

DNA vs. RNA Nucleotides

• DNA: Deoxyribose sugar, bases A, G, C, T

• RNA: Ribose sugar, bases A, G, C, U

DNA and RNA: Structures and Functions

Nucleotide Linkage and Directionality

• Nucleotides are linked by phosphodiester bonds between the 3' hydroxyl of one sugar and the 5' phosphate of the next.

• Polynucleotide chains have directionality: 5' to 3'.

Watson-Crick Base Pairing

• A-T (or A-U in RNA): Two hydrogen bonds

• G-C: Three hydrogen bonds

Major Classes and Functions of RNA

• mRNA: Messenger RNA; carries genetic information from DNA to ribosomes.

• tRNA: Transfer RNA; brings amino acids to the ribosome during translation.

• rRNA: Ribosomal RNA; structural and catalytic component of ribosomes.

RNA Secondary Structures

• Hairpins, loops, bulges, and pseudoknots are common secondary structures formed by base pairing within a single RNA molecule.