Enzymology Lecture Notes PDF

# Enzymology - Naming and enzymes classification - Mechanism of enzyme action - Factors affecting the rate of enzymatic reaction - Inhibition of enzymes - Clinical applications of diagnostic enzymes - Enzymes and biotechnology # Enzymes as Catalysts - Enzymes are proteins that act as catalysts, c...

# Enzymology - Naming and enzymes classification - Mechanism of enzyme action - Factors affecting the rate of enzymatic reaction - Inhibition of enzymes - Clinical applications of diagnostic enzymes - Enzymes and biotechnology # Enzymes as Catalysts - Enzymes are proteins that act as catalysts, compounds that increase the rate of chemical reactions. - Enzymes catalyze the binding of reactants (substrates), converting them to products, and release the products. - Although enzymes may be modified during their participation in this reaction sequence, they return to their original form at the end. # Structure of Enzymes - Enzymes are proteins and, are agreeable to structural analysis by the methods of protein chemistry, molecular biology, and molecular biophysics. - Like all proteins, enzymes are composed mainly of the 20 naturally occurring amino acids. An amino acid is any molecule that conforms at neutral pH to the general formula: $H_3N^+-CH(R)-COO−$ - A diagram showing the structures of the side chains of the 20 naturally occurring amino acids. - **Aliphatic** - Gly - Ala - Val - Leu - Ile - **Hydrophilic** - Ser - Thr - Asp - Glu - Asn - Gln - Lys - Arg - **Sulfur-Containing** - Cys - Met - **Aromatic** - His - Phe - Tyr - Trp - **Other** - Pro # Denaturation of Enzyme - The partial or total alteration of the structure of the enzyme without a change in covalent structure by the action of certain physical procedures (heating, agitation) or chemical agents. - Denaturation can be either reversible or irreversible. # Structural Function Relationship The catalytic activity of an enzyme depends on integrity of the protein structure. This is evidenced by the fact that various factors which disrupt the native conformation of the enzyme polypeptide chain(s) cause loss of the catalytic activity. # Terminologies related to enzymes - **Cofactor:** A non-protein chemical component required for enzyme to initiate its biological activity. The cofactor can be a metal ion (metalloenzymes), or an organic molecule (coenzyme), or a combination of both. - **Apoenzyme:** The protein part of the enzyme without the cofactor necessary for catalysis. - **Holoenzyme:** The active enzyme composed of apoenzyme + cofactor. A diagram showing how:apoenzyme + cofactor -> holoenzyme. # Allosteric enzyme A regulatory enzyme whose affinity for its substrate is affected by the presence or absence of other molecules. i.e. enzyme is altered by combination with a small molecule, referred to as an effector, at a site other than the substrate-binding site, which results in either increased or decreased activity by the enzyme. # Immobilized Enzymes Soluble enzymes bound to an insoluble organic or inorganic matrix or encapsulated within a membrane in order to increase their stability and make possible their repeated or continued use. # Isoenzyme One of a group of related enzymes catalyzing the same reaction but having different molecular structures and characterized by varying physical, biochemical, and immunological properties. # Metal ions as cofactors for specific enzymes (also called metalloenzymes) | Metal | Enzyme | |---|---| | Ca²⁺ | Lipase | | Cu²⁺/Cu⁺ | Cytochrome oxidase, tyrosinase, lysyl/oxidase, superoxide dismutase | | Fe²⁺/Fe³⁺ | Cytochrome oxidase, xanthine oxidase peroxidase, catalase | | K⁺ | Pyruvate kinase | | Mg²⁺ |Hexokinase, phosphofructokinase, enolase, creatine kinase| | Mn²⁺ | Arginase, glycosyl transferase, phosphoglucomutase| | Ni²⁺ | Urease| | Zn²⁺ | Carbonic anhydrase, alkaline phosphatase, DNA polymerase, alcohol dehydrogenase | # Metalloenzymes - The enzymes that require inorganic metal ions as their cofactors are termed metalloenzymes. - Approximately two-third of the known enzymes are metalloenzymes. - In most cases, the binding of metal ion is essentially required for the enzymatic activity (e.g. zinc for carbonic anhydrase), whereas some the enzymes are active even without the metal ion, but its activity increases greatly when the metal ion is added (e.g. chloride and fluoride ions activate salivary amylase). # Coenzyme Many coenzymes are derived from the dietary water-soluble vitamins. **Example:** flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), which are derived from riboflavin (vitamin B2). **In general,** a coenzyme participates in the overall reaction as another substrate, and mainly acts as a donor or acceptor of a chemical group. A table showing coenzymes and the entity transferred | Coenzyme | Entity transferred | |---|---| | Biotin | Carbon dioxide | | Coenzyme A | Acyl group | | FAD | Hydrogen atoms | | NAD⁺ | Hydride ion (H^-) | | Pyridoxal phosphate | Amino groups | | Tetrahydrofolate | One carbon groups (other than CO₂) | | Thiamine pyrophosphate | Hydroxy-ethyl | | Coenzyme B₁₂ | Alkyl groups or hydrogen atoms | # Examples of Coenzymes A diagram showing the structure of: - Nicotinamide adenine dinucleotide (NAD⁺) - Flavin adenine dinucleotide (FAD) # Examples of a biochemical reaction that involved NAD⁺ A diagram showing the conversion of: - Glyceraldehyde 3-phosphate + Inorganic phosphate -> 1,3-Bisphosphoglycerate - NAD⁺ -> NADH + H⁺ # Examples of a biochemical reaction that involved FAD A diagram showing the conversion of: - Succinate + FAD -> Fumarate + FADH₂ # The coenzymes function as either co-substrates or prosthetic groups. ## 1. Co-substrates The co-substrate associates with the active site of the enzyme only transiently for the purpose of the reaction. It is changed chemically during the reaction, and after completion of the reaction, the chemically modified co-substrate dissociates away and is free to participate in other enzymatic reaction cycle. **e.g. pyridoxal phosphate** (a co-substrate in transamination reaction) brings about transfer of amino group from an amino acid to a keto acid by reacting with and serving as transient-carrier of the transferred amino group. A diagram showing the transfer of amino group between: - substrate amino acid (amino donor) -> product amino acid - product keto-acid -> substrate keto-acid (amino acceptor) ## 2. Prosthetic groups The prosthetic group on the other hand is bound permanently to the active site of its enzyme, either covalently or non-covalently. Dissociation of the prosthetic group results in an irreversible loss of catalytic activity of the enzyme. **e.g. Biotin,** is the prosthetic group for a group of enzymes called carboxylase. It is an integral component of these enzymes, being attached by an amide linkage with the apoenzyme component (i.e. apo carboxylase). Dissociation of biotin from the apo carboxylase results in loss of activity of the carboxylase enzymes. **e.g. The most important reaction is the formation of oxaloacetate from pyruvate by pyruvate carboxylase enzyme in presence of Biotin.** A diagram showing the conversion of: - pyruvate + CO₂ -> oxaloacetate # Specificity of Enzymes - Enzymes are the most remarkable and highly specialized proteins, they have a high degree of specificity for their substrates, and they accelerate chemical reactions tremendously. In general, four types of behavior can be described: 1. Absolute specificity - catalyze only one reaction. 2. Group specificity - catalyze a functional group, which can occur in a variety of substrate. 3. Linkage specificity - catalyze a chemical bond regardless of the rest of the molecular structure. 4. Stereochemical specificity - the enzyme will act on a steric or optical isomer. # Sources of Enzymes - Enzymes occur in all living organisms and catalyze biochemical reactions necessary to support life. A wide array of enzymes is extracted from plant sources; they have many advantages including cost of production and stability of products. **Microbes are preferred to plants and animals as sources of enzymes because:** ○ They are generally cheaper to produce. ○ **Regular supply** due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media. ○ Microbial enzymes are also more stable than their corresponding plant and animal enzymes. ○ Plant and animal tissues contain **more potentially harmful materials than microbes**, including phenolic compounds (from plants), endogenous enzyme inhibitors and proteases.