enzymes_handouts
Introduction
Catalysts: Substances that increase the velocity or rate of a chemical reaction without undergoing any change in overall process.
Enzymes: Defined as biocatalysts synthesized by living cells, protein in nature, colloidal and thermolabile, specific in action.
Historical Background
1836: Berzelius coined the term "catalysis."
Kuhne: Used the word "enzyme" for catalysis in biological systems.
1883: Buchner isolated enzymes from cell-free yeast extract.
1926: James Sumner isolated and crystallized urease from Jack bean, identifying it as a protein.
Nomenclature and Classification
Naming Enzymes: Suffix "-ase" is added to the substrate name.
IUB System: Enzyme classification started in 1964; divided into six major classes.
IUB System of Classification
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
Oxidoreductases
Involved in oxidation-reduction reactions.
Examples: Lactate dehydrogenase, alcohol dehydrogenase.
Reaction: AH2 + B → A + BH2 (oxidation-reduction).
Transferases
Catalyze the transfer of functional groups.
Examples: Transaminases, hexokinase.
Reaction: A-X + B → A + B-X (group transfer).
Hydrolases
Bring about hydrolysis of various compounds.
Examples: Lipase, pepsin, urease.
Reaction: A-B + H2O → AH + BOH (hydrolysis).
Lyases
Specialize in addition or removal of water, ammonia, etc.
Examples: Aldolase, fumarase.
Reaction: A-B + X-Y → AX-BY (addition-elimination).
Isomerases
Involved in isomerization reactions.
Examples: Phosphohexose isomerase, retinol isomerase.
Reaction: A → A’ (interconversion of isomers).
Ligases
Catalyze synthetic reactions.
Examples: Succinate thiokinase, glutamine synthetase.
Reaction: A + B → A-B + ATP (condensation).
Enzyme Commission Number (EC)
Subclassification of enzymes with a four-digit EC number.
Represents:
Class (First digit)
Sub-class (Second digit)
Sub-sub class (Third digit)
Individual enzyme (Fourth digit)
Example: EC 1.1.1.1 for alcohol dehydrogenase.
Characteristics of Enzymes
Most enzymes are proteins, heat-labile, and water-soluble.
Can be precipitated by reagents like ammonium sulfate.
Contains about 16% nitrogen by weight.
Chemical Nature of Enzymes
All enzymes are proteins, except for RNA acting as ribozyme.
Each enzyme has its own tertiary structure and conformation.
Can be entirely protein or contain a non-protein part (holoenzyme = apoenzyme + coenzyme).
Prosthetic Groups
Non-protein part tightly bound to the enzyme.
Examples: Zn in carbonic anhydrase, Iron in cytochrome oxidase.
Ribozyme & Abzyme
Ribozymes: RNA molecules with catalytic activity.
Abzymes: Catalytic antibodies or antibody enzymes.
Properties of Enzymes
Active Sites
contain amino acid side chains for substrate binding and catalysis.
Formation of enzyme-substrate (ES) complexes occurring at active sites.
Catalytic Efficiency
Enzymes can enhance reaction speed 10^3 to 10^8 times.
Turnover number (Kcat) is the number of substrate molecules converted to product per enzyme per second (typically 10^2 to 10^4 s^-1).
Catalytic efficiency calculated by Kcat/Km.
Specificity
Ability to select specific substrates from a set of similar molecules.
Types of Specificity:
Bond Specificity: Specific to bond types.
Group Specificity: Specific to bonds and surrounding groups.
Substrate Specificity: Specific to one substrate.
Stereospecificity: Specific to optical isomers.
Geometrical Specificity: Specific to similar geometries.
Cofactor Specificity: Specific to substrate-cofactor combinations.
Regulation of Enzyme Activity
Enzymatic activity can be increased or decreased based on cellular needs.
Location in Cells
Enzymes often localized in specific organelles to isolate substrates/products from other reactions.
Mechanism of Enzyme Actions
Enzymatic reaction involves formation of ES complexes leading to product formation.
Enzymes lower activation energy required for reactions.
They provide a scenario where substrates are put into a higher energy state to facilitate transformation.
Enzyme-Substrate Complex Formation
Substrate must bind to enzyme at active site to form an ES complex, resulting in product (P) formation:
E + S ⇌ ES → E + P
Models of Enzyme Function
Lock and Key Model
Proposed by Emil Fischer; suggests rigid structure of enzyme with a specific active site.
Lacks the explanation for flexibility in enzyme activity.
Induced Fit Theory
Proposed by Koshland (1958); suggests that substrate induces a conformational change in the enzyme upon binding.
Mechanisms of Enzyme Catalysis
Types of Catalysis:
Catalysis by Proximity: Increases likelihood of molecular interaction by reducing the distance between substrates.
Acid-Base Catalysis: Amino acids act as acids or bases, assisting substrate transformations.
Catalysis by Strain: Stretches or distorts bonds, making them vulnerable to cleavage (e.g., lysozyme).
Covalent Catalysis: Formation of transient covalent bonds between enzyme and substrate.
Enzyme Kinetics
Kinetic analysis characterizes enzyme actions even before isolation.
Michaelis-Menten equation expresses the relationship between [S] and rate of reaction (V0).
The reaction rate is influenced by substrate concentration based on whether [S] is low or high relative to Km.
Lineweaver-Burk Plot
A method to determine Km and Vmax by plotting reciprocal rates versus substrate concentrations, yielding a straight line for analysis.
Slope: Km/Vmax, Y-intercept: 1/Vmax.
Factors Affecting Enzyme Activity
Concentration of Enzyme: Increasing enzyme concentration typically increases reaction velocity.
Concentration of Substrate: Increasing substrate concentration increases reaction velocity up to a limit.
Temperature: Bell-shaped effect; optimum activity typically 35°C–40°C.
pH: Bell-shaped effect; each enzyme has an optimum pH.
Product Concentration: Accumulation of products decreases velocity.
Activators: Enhance enzyme activity (e.g., chloride ions for amylase).
Units of Enzyme Activity
Enzyme Unit (U): Amount of enzyme converting one micromole of substrate per minute under specified conditions.
Katal: SI unit defined as the conversion of one mole of substrate per second.