6.3 Enzymes

Enzymes are protein catalysts that speed up chemical reactions in living organisms. They play crucial roles in metabolism, digestion, and cellular signaling. Understanding enzyme structure, function, and regulation is key to developing targeted therapies in medicinal chemistry.

This topic covers enzyme structure, enzyme-substrate interactions, kinetics, inhibition, and regulation. It also explores coenzymes, enzyme classification, drug targeting, immobilization, and the role of enzymes in disease diagnosis and treatment. These concepts are fundamental to understanding enzyme function in biological systems.

Enzymes as biological catalysts

• Enzymes are proteins that act as biological catalysts, speeding up chemical reactions in living organisms
• They play a crucial role in various biochemical processes, including metabolism, digestion, and cellular signaling
• Understanding the structure, function, and regulation of enzymes is essential for developing targeted therapies and drugs in medicinal chemistry

Structure of enzymes

Amino acid composition

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• Enzymes are composed of amino acids linked together by peptide bonds
• The specific sequence of amino acids determines the unique structure and function of each enzyme
• The amino acid composition influences the enzyme's stability, solubility, and catalytic activity

Primary, secondary, tertiary structure

• The primary structure refers to the linear sequence of amino acids in the polypeptide chain
• Secondary structure involves local folding patterns, such as α-helices and β-sheets, stabilized by hydrogen bonds
• Tertiary structure is the three-dimensional shape of the enzyme, resulting from interactions between amino acid side chains (disulfide bonds, hydrophobic interactions, etc.)

Active site for substrate binding

• The active site is a specific region of the enzyme where the substrate binds and the catalytic reaction occurs
• It is typically a cleft or pocket formed by the tertiary structure of the enzyme
• The active site contains amino acid residues that interact with the substrate and facilitate the chemical reaction

Enzyme-substrate interactions

Lock and key model

• The lock and key model proposes that the active site of an enzyme has a rigid, complementary shape to the substrate
• The substrate fits precisely into the active site, like a key fitting into a lock
• This model explains the specificity of enzymes but does not account for the flexibility of the active site

Induced fit model

• The induced fit model suggests that the active site of an enzyme is flexible and can change shape upon substrate binding
• The initial interaction between the enzyme and substrate induces a conformational change in the enzyme, optimizing the active site for catalysis
• This model better explains the observed flexibility and adaptability of enzymes

Substrate specificity of enzymes

• Enzymes exhibit high specificity for their substrates due to the unique structure of their active sites
• The specificity is determined by the shape, size, and chemical properties of the active site
• Substrate specificity ensures that enzymes catalyze specific reactions without interfering with other cellular processes

Enzyme kinetics

Factors affecting reaction rates

• Temperature: Increasing temperature generally increases reaction rates until the enzyme denatures
• pH: Enzymes have an optimal pH range where they function most efficiently; extreme pH can denature the enzyme
• Substrate concentration: Increasing substrate concentration increases reaction rates until the enzyme becomes saturated

Michaelis-Menten equation

• The Michaelis-Menten equation describes the relationship between reaction rate (v) and substrate concentration (S): $v = \frac{V_{max}[S]}{K_m + [S]}$
• It assumes steady-state conditions and a single substrate
• The equation helps determine important kinetic parameters, such as $K_m$ and $V_{max}$

Km and Vmax parameters

• $K_m$ (Michaelis constant) is the substrate concentration at which the reaction rate is half of $V_{max}$
• It indicates the affinity of the enzyme for the substrate; a lower $K_m$ value suggests higher affinity
• $V_{max}$ is the maximum reaction rate achieved when the enzyme is saturated with substrate
• $V_{max}$ depends on the enzyme concentration and the catalytic efficiency of the enzyme

Enzyme inhibition

Competitive vs noncompetitive inhibition

• Competitive inhibition occurs when the inhibitor binds to the active site, competing with the substrate
• The inhibitor structurally resembles the substrate and reduces the apparent affinity of the enzyme for the substrate
• Noncompetitive inhibition involves the inhibitor binding to a site other than the active site, causing a conformational change that reduces the enzyme's activity

Reversible vs irreversible inhibitors

• Reversible inhibitors bind non-covalently to the enzyme and can be removed by dilution or dialysis
• They include competitive, noncompetitive, and uncompetitive inhibitors
• Irreversible inhibitors form covalent bonds with the enzyme, permanently inactivating it
• They are often used as drugs to target specific enzymes involved in disease processes

Inhibition constants (Ki)

• The inhibition constant ($K_i$) is a measure of the potency of an inhibitor
• It represents the concentration of the inhibitor required to reduce the enzyme activity by half
• A lower $K_i$ value indicates a more potent inhibitor, as it requires less inhibitor to achieve the same level of inhibition

Regulation of enzyme activity

Allosteric regulation

• Allosteric regulation involves the binding of effector molecules to sites other than the active site (allosteric sites)
• Allosteric effectors can be activators or inhibitors, modulating the enzyme's activity by inducing conformational changes
• This type of regulation allows for fine-tuning of enzyme activity in response to cellular conditions

Covalent modifications

• Enzymes can be regulated by covalent modifications, such as phosphorylation, acetylation, or glycosylation
• These modifications can alter the enzyme's structure, stability, or activity
• Kinases and phosphatases are examples of enzymes that add or remove phosphate groups, respectively, to regulate other enzymes

Zymogen activation

• Zymogens are inactive precursors of enzymes that require proteolytic cleavage for activation
• This mechanism ensures that enzymes are produced in an inactive form and activated only when needed
• Examples include digestive enzymes (pepsinogen, trypsinogen) and blood clotting factors (prothrombin)

Coenzymes and cofactors

Role in enzyme catalysis

• Coenzymes and cofactors are non-protein molecules that assist enzymes in catalyzing reactions
• They can act as electron carriers, group transfer agents, or structural components of the enzyme
• Many coenzymes are derived from vitamins, highlighting the importance of dietary factors in enzyme function

Common examples (NAD+, FAD, etc.)

• Nicotinamide adenine dinucleotide (NAD+) and its phosphorylated form (NADP+) are involved in redox reactions
• Flavin adenine dinucleotide (FAD) is another redox coenzyme derived from riboflavin (vitamin B2)
• Coenzyme A (CoA) is essential for the transfer of acyl groups in metabolic pathways

Vitamin-derived coenzymes

• Thiamine pyrophosphate (TPP), derived from vitamin B1, is a cofactor for decarboxylation reactions
• Pyridoxal phosphate (PLP), derived from vitamin B6, is involved in amino acid metabolism
• Biotin, a B-vitamin, acts as a carrier of carboxyl groups in carboxylation reactions

Enzyme classification

International enzyme nomenclature

• The International Union of Biochemistry and Molecular Biology (IUBMB) has established a standardized system for naming and classifying enzymes
• Enzymes are named according to the reaction they catalyze, with the suffix "-ase" added to the substrate or type of reaction

Six main enzyme classes

• Oxidoreductases: catalyze redox reactions, transferring electrons between molecules
• Transferases: transfer functional groups (methyl, acyl, phosphoryl, etc.) from one molecule to another
• Hydrolases: catalyze the hydrolysis of chemical bonds, such as esters, glycosides, or peptides
• Lyases: catalyze non-hydrolytic addition or removal of groups from substrates, often forming double bonds
• Isomerases: catalyze the rearrangement of atoms within a molecule, resulting in isomeric forms
• Ligases: catalyze the joining of two molecules, typically coupled with the hydrolysis of ATP

EC numbering system

• The Enzyme Commission (EC) number is a numerical classification scheme for enzymes
• Each enzyme is assigned a four-digit number: EC x.y.z.w
• The first digit (x) represents the main enzyme class (1-6)
• The second digit (y) indicates the subclass, the third digit (z) denotes the sub-subclass, and the fourth digit (w) is the serial number of the enzyme within its sub-subclass

Enzymes as drug targets

Rational drug design strategies

• Enzymes are attractive targets for drug development due to their involvement in various disease processes
• Structure-based drug design involves analyzing the 3D structure of the enzyme to design molecules that can bind and modulate its activity
• Ligand-based drug design uses known inhibitors or substrates as templates to develop new drugs with improved properties

Examples of enzyme-targeted drugs

• Statins (atorvastatin, simvastatin) inhibit HMG-CoA reductase, an enzyme involved in cholesterol biosynthesis, to treat hypercholesterolemia
• Angiotensin-converting enzyme (ACE) inhibitors (captopril, enalapril) target ACE to treat hypertension and heart failure
• Protease inhibitors (saquinavir, ritonavir) target viral proteases to treat HIV infection

Advantages and challenges

• Targeting enzymes allows for the development of specific and potent drugs with fewer side effects
• Enzyme inhibitors can be designed to have high selectivity for the target enzyme over related enzymes
• Challenges include the potential for drug resistance, especially with rapidly mutating targets (viral enzymes)
• Enzyme redundancy and compensatory mechanisms in the cell can sometimes limit the effectiveness of enzyme-targeted drugs

Enzyme immobilization

Methods for enzyme immobilization

• Adsorption: enzymes are physically adsorbed onto a solid support material through weak interactions
• Covalent bonding: enzymes are chemically attached to a support material via covalent bonds
• Entrapment: enzymes are physically trapped within a polymeric matrix or gel
• Cross-linking: enzymes are directly cross-linked with each other or with a support material using bifunctional reagents

Applications in industry and medicine

• Immobilized enzymes are used in the production of high-fructose corn syrup, using glucose isomerase
• Lactose-free milk is produced using immobilized lactase to hydrolyze lactose
• Immobilized enzymes are used in biosensors for the detection of glucose, urea, or other analytes
• Enzyme-based bioreactors are used for the treatment of industrial waste and pollutants

Improved stability and reusability

• Immobilization enhances the stability of enzymes by protecting them from denaturation and aggregation
• Immobilized enzymes can be easily separated from the reaction mixture and reused multiple times
• The improved stability and reusability of immobilized enzymes make them cost-effective for industrial applications

Enzymes in disease and diagnosis

Enzyme deficiencies and disorders

• Inborn errors of metabolism are genetic disorders caused by deficiencies in specific enzymes
• Examples include phenylketonuria (PKU), caused by a deficiency in phenylalanine hydroxylase, and galactosemia, caused by a deficiency in galactose-1-phosphate uridyltransferase
• Enzyme deficiencies can lead to the accumulation of toxic substrates or the lack of essential products, causing various symptoms

Diagnostic enzyme assays

• Enzyme assays are used to diagnose diseases by measuring the activity of specific enzymes in biological samples (blood, urine, etc.)
• Elevated levels of liver enzymes (ALT, AST) can indicate liver damage or disease
• Creatine kinase (CK) levels are used to diagnose muscle damage, such as in heart attacks or muscular dystrophy

Enzyme replacement therapy

• Enzyme replacement therapy involves administering a functional version of the deficient enzyme to treat the disorder
• Examples include recombinant glucocerebrosidase for Gaucher disease and recombinant α-galactosidase A for Fabry disease
• Challenges include the potential for immune reactions, the need for repeated administrations, and the high cost of production