Biotechnology: Class 11 — Chapter 4: Enzymes and Bioenergetics

• Molecular Weight: Ranges from approximately 2000 to over one million Daltons.
• Holoenzyme: The complete, catalytically active enzyme system consisting of the protein component (apoenzyme) and its non-protein component (cofactor).
• Coenzyme: A complex organic cofactor, typically derived from vitamins, that transiently participates in catalysis as a carrier of specific functional groups.
• Prosthetic Group: A coenzyme or metal ion cofactor that is tightly and covalently bound to the apoenzyme.

Table 4.1: Coenzymes, Precursor Vitamins, and Catalytic Roles

Table 4.2: Metal Ions Serving as Cofactors

Adopted by the International Union of Biochemistry (I.U.B.) in 1964, enzymes are classified into seven major classes based on the type of reaction they catalyze.

Table 4.3: I.U.B. Enzyme Classification

• Isozymes (Isoenzymes): Multiple molecular forms of the same enzyme occurring within the same species, tissue, or cell. They catalyze the identical reaction but differ in amino acid composition, physical properties, and kinetic parameters. Examples include hexokinase (4 forms) and lactate dehydrogenase (LDH) (5 forms in humans).
• Enzyme Active Site: A highly defined, three-dimensional pocket or cleft formed by the folding of the polypeptide chain. It binds the substrate through weak, non-covalent interactions including electrostatic forces, hydrogen bonds, Van der Waals interactions, and hydrophobic forces.

1. Fischer's Lock and Key Model (1894): Proposes that the active site of the free enzyme is rigid and pre-shaped to perfectly match the complementary structure of the substrate, analogous to a key fitting into a lock.
2. Koshland's Induced Fit Model (1958): Proposes that the active site is flexible. Binding of the substrate induces a conformational change in the enzyme, aligning catalytic groups into the correct orientation for reaction.

• Group Specificity: Enzyme acts on structurally related substrates sharing a common functional group.
• Absolute Specificity: Enzyme acts exclusively on one single substrate.
• Stereochemical (Optical) Specificity: Enzyme distinguishes between optical isomers (e.g., D-amino acid oxidase acts only on D-amino acids).
• Geometrical Specificity: Enzyme is selective for cis or trans isomers (e.g., fumarase interconverts fumarate and malate).

• Temperature: Reaction rate increases with temperature up to an optimum (typically 40-45°C for most enzymes; 37°C in humans), beyond which thermal denaturation occurs. Exceptional enzymes like Taq DNA polymerase remain active at 100°C.
• pH: Most enzymes exhibit a bell-shaped activity curve with a distinct optimum pH. Extremes of pH alter ionization states of active site residues. Exceptions include pepsin (optimum pH 1-2) and alkaline phosphatase (optimum pH 10-11).
• Substrate Concentration: Rate increases proportionally with substrate concentration $[S]$ until the enzyme becomes saturated, reaching a plateau at maximum velocity ($V_{max}$).

• Enzyme Unit (U): The amount of enzyme that catalyzes the conversion of 1 micromole ($\mu\text{mol}$) of substrate per minute under standard conditions.
• Katal (kat): The SI unit of catalytic activity, defined as the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second ($1\text{ kat} = 6 \times 10^7\text{ U}$).
• Specific Activity: The units of enzyme activity per milligram of total protein, serving as a measure of enzyme purity.

Enzymes accelerate chemical reactions by lowering the activation energy ($\Delta G^A$) required to reach the high-energy transition state. They do not alter the overall free energy change ($\Delta G$) or the equilibrium constant of the reaction; they only accelerate the rate at which equilibrium is attained.

The kinetics of single-substrate enzyme-catalyzed reactions are described by the Michaelis-Menten equation:

$$v_0 = \frac{V_{max}[S]}{K_m + [S]}$$

• $v_0$: Initial velocity of the reaction.
• $V_{max}$: Maximum velocity achieved at saturating substrate concentrations.
• $K_m$ (Michaelis Constant): The substrate concentration at which the reaction rate is half of $V_{max}$. It is an intrinsic property of the enzyme reflecting its affinity for the substrate (lower $K_m$ indicates higher affinity).

• Irreversible Inhibition: Inhibitor binds covalently or extremely tightly to the enzyme, permanently inactivating it. Examples include penicillin (inhibits transpeptidase) and aspirin (inhibits cyclooxygenase).
• Reversible Inhibition: Inhibitor dissociates rapidly from the enzyme-inhibitor complex. It is classified into three primary types:

1. Competitive Inhibition: Inhibitor structurally resembles the substrate and competes for the active site. It increases the apparent $K_m$ ($K'_m > K_m$) but does not affect $V_{max}$. Can be overcome by increasing $[S]$.
2. Non-Competitive Inhibition: Inhibitor binds to a site distinct from the active site, on both free enzyme and ES complex. It decreases $V_{max}$ but does not alter $K_m$. Cannot be overcome by increasing $[S]$.
3. Uncompetitive Inhibition: Inhibitor binds exclusively to the enzyme-substrate (ES) complex. Both $K_m$ and $V_{max}$ are decreased.

Allosteric enzymes do not obey classical Michaelis-Menten kinetics and yield a sigmoidal (S-shaped) curve when plotting $v_0$ against $[S]$. They possess distinct regulatory (allosteric) sites in addition to active sites. Binding of effector molecules to these regulatory sites reversibly alters the enzyme's affinity for the substrate, serving as key control points in metabolic pathways.

States that energy can neither be created nor destroyed, only converted from one form to another. The total energy of the universe remains constant. Mathematically expressed as:

$$\Delta E = E_B - E_A = Q - W$$

Where $\Delta E$ is the change in internal energy, $Q$ is heat absorbed, and $W$ is work done by the system.

States that the physical universe continuously moves toward a state of maximum disorder, meaning the total entropy ($S$) of the universe is always increasing. For any spontaneous process:

$$\Delta S_{system} + \Delta S_{surroundings} > 0$$

Living organisms maintain highly ordered, low-entropy states by consuming external chemical or light energy, releasing heat and disordered byproducts into their surroundings.

Combining the first and second laws yields the Gibbs free energy equation, which determines reaction spontaneity at constant temperature and pressure:

$$\Delta G = \Delta H - T\Delta S$$

• $\Delta G$: Change in free energy (energy available to do useful work). If $\Delta G < 0$, the reaction is spontaneous (exergonic). If $\Delta G > 0$, the reaction requires energy input (endergonic).
• $\Delta H$: Change in enthalpy (heat content).
• $T\Delta S$: Temperature-entropy factor (energy unavailable to do work).

• Chemical Composition: Composed of an adenine purine base, a ribose pentose sugar, and three phosphate groups linked in series (alpha, beta, and gamma).
• Energy Release: Hydrolysis of the terminal phosphoanhydride bond of ATP to form ADP and inorganic phosphate ($P_i$) releases approximately $7.3\text{ kcal/mol}$ ($30.5\text{ kJ/mol}$) of free energy under standard conditions:

$$\text{ATP} + \text{H}_2\text{O} \rightarrow \text{ADP} + \text{P}_i \quad (\Delta G^{\circ\prime} = -7.3\text{ kcal/mol})$$

• AMP Formation: In certain high-energy reactions, ATP is cleaved into adenosine monophosphate (AMP) and pyrophosphate ($PP_i$).