Comprehensive Study Guide on Enzymes and Coenzymes

General Characteristics of Enzymes

• Enzyme Definition: Usually a protein that acts as a catalyst in a specific biochemical reaction.

• Abundance: Every cell in the human body contains thousands of different enzymes.

• Specificity: Every single reaction in a cell requires its own specific enzyme.

• Composition: Most enzymes are globular proteins. However, a few enzymes are made of RNA, which catalyze biochemical reactions involving nucleic acids.

• Denaturation: Enzymes undergo all the typical reactions of proteins, including denaturation due to changes in pH or temperature.

  • For example, a person suffering from a high fever runs the risk of denaturing certain enzymes.

Enzyme Structure and Components

• Simple Enzymes: These are composed entirely of protein.

• Conjugated Enzymes: These consist of a protein part and a non-protein part.

  • Apoenzyme: The protein-only part of a conjugated enzyme. It is not functional by itself.

  • Cofactor (Coenzyme): The non-protein part of a conjugated enzyme.

    • An apoenzyme cannot catalyze its reaction without its specific cofactor.

  • Holoenzyme: The combination of the apoenzyme and the cofactor ($\text{Holoenzyme} = \text{Apoenzyme} + \text{Cofactor}$). This is the biochemically active, functional conjugated enzyme.

• Nature of Coenzymes and Cofactors:

  • They provide additional chemically reactive functional groups beyond those present in amino acids.

  • They include small organic molecules or inorganic ions.

  • Metal Ions: Often act as cofactors; examples include $Zn^{2+}$, $Mg^{2+}$, $Mn^{2+}$, and $Fe^{2+}$. These can be bound directly to the enzyme or to a coenzyme.

  • Coenzyme: A small organic molecule acting as a cofactor. Many are derived from vitamins, especially B-vitamins (e.g., $NAD^+$).

Enzyme Nomenclature and Classification

• Naming Conventions: Enzymes are named based on the type of reaction they catalyze and/or their substrate.

• Substrate: The reactant upon which the specific enzyme acts. The enzyme physically binds to the substrate to form an Enzyme/Substrate complex.

• Suffixes:

  • -ase: The standard suffix denoting an enzyme (e.g., lactase, amylase, lipase, protease).

  • -in: Found in some of the first digestive enzymes studied (e.g., pepsin, trypsin, chymotrypsin).

• Prefixes: Often denote the reaction type (e.g., Oxidase for redox reactions; Hydrolase for adding water to break a component).

• Combination Names: Substrate identity and reaction type are often combined (e.g., Pyruvate carboxylase, Lactate dehydrogenase).

• Six Major Classes of Enzymes:

  1. Oxidoreductase: Catalyzes redox reactions (oxidation and reduction). Example: Dehydrogenases.

  2. Transferase: Catalyzes the transfer of a functional group from one molecule to another. Examples: Transaminases (transfer amino groups) and Kinases (transfer phosphate groups).

  3. Hydrolase: Catalyzes hydrolysis reactions. Examples: Lipases (lipid hydrolysis) and Proteases (protein hydrolysis).

  4. Lyase: Catalyzes the addition or removal of atoms to or from a double bond. Example: Decarboxylases (removal of carboxyl groups).

  5. Isomerase: Catalyzes isomerization reactions. Examples: Isomerases (aldose to ketose conversion) and Mutases (transfer of a functional group within a substrate).

  6. Ligase: Catalyzes synthesis reactions, joining two molecules into one and forming a new chemical bond, coupled with ATP hydrolysis. Example: Synthetases.

Mechanisms of Enzyme Action

• Active Site: The specific 3-D 'crevice-like' cavity or binding cleft where the substrate binds. It is formed by the folding of the protein (secondary and tertiary structures). An enzyme can have more than one active site. The R-groups of amino acids in this site determine substrate specificity.

• Enzyme-Substrate Complex: Formed temporarily when the substrate binds to the active site. This lowers activation energy, allowing the reaction to occur much faster.

• Lock and Key Model: The active site has a fixed, rigid shape (the lock). The substrate (the key) must fit exactly and maintain a complementary geometry.

• Induced Fit Model: The enzyme is flexible and constantly changes shape. The active site adjusts to accommodate and accept the substrate. Analogy: A glove (enzyme) changing shape when a hand (substrate) is inserted.

Enzyme Specificity

• Absolute Specificity: The enzyme catalyzes a reaction for only one specific substrate. This is the most restrictive type. Examples: Catalase for hydrogen peroxide ($H_2O_2$); Urease for urea.

• Group Specificity: The enzyme acts only on similar substrates with a specific functional group. Examples: Carboxypeptidase (cleaves amino acids from the carboxyl end); Hexokinase (adds phosphate to hexoses).

• Linkage Specificity: The enzyme acts on a particular type of chemical bond regardless of the rest of the structure. This is the most general type. Examples: Phosphatases (hydrolyze phosphate-ester bonds); Chymotrypsin (hydrolyzes peptide bonds).

• Stereochemical Specificity: The enzyme distinguishes between stereoisomers due to the inherent chirality of the active site. Example: L-Amino-acid oxidase acts on L-amino acids but not D-amino acids.

Factors Affecting Enzyme Activity

• Enzyme Activity Definition: A measure of the rate at which an enzyme converts substrate to products.

• Temperature ($t$):

  • Increased $t$ lead to increased kinetic energy, more collisions, and a higher reaction rate.

  • Optimum Temperature ($t_{OPT}$): The temperature of maximum activity. For human enzymes, $t_{OPT} = 37^{\circ}C$.

  • Beyond $t_{OPT}$, the tertiary structure changes, leading to inactivation and denaturation.

• pH:

  • Optimum pH ($pH_{OPT}$): The pH where activity is maximized. Most enzymes have a narrow range (usually $7.0 - 7.5$).

  • Exceptions: Pepsin (stomach) has $pH_{OPT} = 2.0$; Trypsin (small intestine) has $pH_{OPT} = 8.0$.

• Substrate Concentration ($[substrate]$):

  • With constant $[enzyme]$, increasing $[substrate]$ increases the rate until saturation occurs.

  • At saturation, all active sites are occupied, and the rate levels off. The enzyme is at maximum capacity.

• Enzyme Concentration ($[enzyme]$):

  • Reaction rate is directly proportional to the amount of enzyme present, provided $[substrate]$ is constant and in excess.

  • In living cells, $[substrate]$ is typically much higher than $[enzyme]$. Enzymes are not consumed and are reused multiple times.

Enzyme Inhibition

• Reversible Competitive Inhibition: The inhibitor resembles the substrate and competes for the active site. If the inhibitor binds, the substrate cannot. This is reversed by significantly increasing the $[substrate]$. Example: Anti-histamines inhibit histidine decarboxylase.

• Reversible Non-competitive Inhibition: The inhibitor binds to a site other than the active site, altering the 3-D structure of the enzyme and the active site so the substrate cannot fit. This is reversed by lowering the $[inhibitor]$. Example: Heavy metals like $Pb^{2+}$ and $Hg^{2+}$ binding to Cysteine $-SH$ groups.

• Irreversible Inhibition: The inhibitor forms a strong covalent bond to the active site, permanently deactivating the enzyme. These do not resemble the substrate. Example: Nerve gases in chemical warfare and organophosphate insecticides.

Regulation of Enzyme Activity

• Allosteric Enzymes: These have quaternary structures with two or more binding sites.

  • Active Site: Site for the substrate.

  • Regulatory Site: Site for a regulator molecule.

  • Positive Regulator: Up-regulates activity by enhancing the active site.

  • Negative Regulator: Down-regulates activity (non-competitive inhibition) by compromising the active site.

• Feedback Control: A process where the product of a reaction sequence inhibits or activates an earlier step. Example: Phosphofructokinase (PFK) in glycolysis is allosterically inhibited by ATP when cellular ATP is in excess.

• Proteolytic Enzymes and Zymogens: Enzymes produced in an inactive form (zymogens or proenzymes) to prevent them from destroying the tissues that produce them.

  • Activation requires removing a peptide fragment to change the 3-D shape. Example: Pepsinogen (inactive) becomes Pepsin (active).

• Covalent Modification: Altering activity by adding or removing groups.

  • Phosphorylation: Addition of a phosphate group (from ATP) via a Kinase enzyme.

  • Dephosphorylation: Removal of a phosphate group via a Phosphatase enzyme.

  • Examples: Glycogen synthase is deactivated by phosphorylation; Glycogen phosphorylase is activated by phosphorylation.

Medical and Pharmacological Applications

• Drug Inhibitors:

  • ACE Inhibitors: Inhibit Angiotensin-Converting Enzyme to lower blood pressure.

  • Sulfa Drugs: Act as competitive inhibitors for bacterial enzymes converting PABA to Folic acid, retarding bacterial growth.

  • Penicillins: $\beta$-lactam antibiotics that inhibit transpeptidase, preventing the formation of cross-links in bacterial cell walls, leading to cell death.

• Diagnostic Enzymes:

  • Lactate Dehydrogenase (LDH): Elevated blood levels indicate myocardial infarction (MI) or liver disease.

  • Tissue Plasminogen Activator (TPA): Activates plasminogen to dissolve clots during MI treatment.

  • Urease: Used as an indicator for urea in Blood Urea Nitrogen (BUN) tests to assess kidney function.

• Isoenzymes: Enzymes that catalyze the same reaction but in different tissues (markers for location).

  • LDH has 5 isoenzymes. $LDH1$ is prevalent in heart muscle (its presence in blood indicates heart damage), while $LDH5$ is found in skeletal muscle and liver.

• Blood Enzyme Assay Indicators:

  • CPK: Heart disease.

  • AST / ALT: Heart disease, liver disease, or muscle damage.

  • GGTP: Heart disease, liver disease.

  • ALP: Bone or liver disease.

Essential Vitamins as Coenzymes

Questions and Discussion

• Question: What is the function of an enzyme in a chemical reaction?

  • Response: It acts as a catalyst to facilitate and speed up specific biochemical reactions.

• Question: What happens to the enzymes when the body temperature rises from $37^{\circ}C$ to $42^{\circ}C$?

  • Response: The enzymes risk denaturation; their tertiary structure changes, causing them to lose function.

• Question: If an enzyme has broken down and is non-functional, what would happen to the chemical reaction normally facilitated by the enzyme?

  • Response: The reaction would slow down significantly or stop entirely because the activation energy barrier would be too high to overcome without the catalyst.