Enzymology Basics

Cheatsheet Content

### Enzymes: Definition Enzymes are biological catalysts, primarily proteins, that accelerate the rate of biochemical reactions without being consumed in the process. They are highly specific, acting on particular substrates to produce specific products. ### Important Terms - **Enzyme Unit (U):** One international unit (IU or U) of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 micromole ($\mu$mol) of substrate per minute under specified conditions. - **Katal (kat):** The SI unit of enzyme activity, defined as the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second. $1 \text{ kat} = 60 \times 10^6 \text{ U}$. - **Specific Activity:** The number of enzymatic units per milligram of total protein (U/mg protein). It measures the purity of an enzyme. - **Turnover Number ($k_{cat}$):** The maximum number of substrate molecules converted to product per enzyme molecule per unit of time, when the enzyme is saturated with substrate. ### Classification of Enzymes Enzymes are classified into six major classes by the International Union of Biochemistry and Molecular Biology (IUBMB) based on the type of reaction they catalyze: 1. **Oxidoreductases:** Catalyze oxidation-reduction reactions (transfer of electrons or hydrogen atoms). * *Example:* Dehydrogenases, oxidases. 2. **Transferases:** Catalyze the transfer of a functional group (e.g., methyl, phosphate, amino) from one molecule to another. * *Example:* Kinases, transaminases. 3. **Hydrolases:** Catalyze the hydrolytic cleavage of C-O, C-N, C-C, and other bonds by adding water. * *Example:* Lipases, proteases, nucleases. 4. **Lyases:** Catalyze the cleavage of C-C, C-O, C-N, and other bonds by elimination, often forming double bonds or rings; also catalyze the addition of groups to double bonds. * *Example:* Decarboxylases, aldolases. 5. **Isomerases:** Catalyze the rearrangement of atoms within a molecule, converting one isomer to another. * *Example:* Racemases, mutases, epimerases. 6. **Ligases:** Catalyze the joining of two molecules with the concomitant hydrolysis of ATP or another energy-rich compound. * *Example:* Synthetases, carboxylases. ### Physico-Chemical Properties - **Protein Nature:** Most enzymes are globular proteins, giving them specific three-dimensional structures essential for their function. - **Specificity:** Enzymes exhibit high specificity: - **Substrate Specificity:** Act on specific substrates (e.g., absolute, group, stereochemical). - **Reaction Specificity:** Catalyze only one type of reaction. - **Catalytic Efficiency:** Can increase reaction rates by factors of $10^5$ to $10^{17}$ compared to uncatalyzed reactions. - **Thermolability:** Generally sensitive to temperature changes; activity increases with temperature to an optimum, then rapidly decreases due to denaturation. - **pH Sensitivity:** Have an optimal pH range for activity; deviations from this range can lead to denaturation and loss of activity. ### Factors Affecting Activity - **Temperature:** - Low temperatures: Decrease kinetic energy, slowing reaction. - Optimum temperature: Maximal activity. - High temperatures: Cause irreversible denaturation of the enzyme's 3D structure, leading to loss of activity. - **pH:** - Optimum pH: Specific pH at which enzyme activity is maximal. - Extreme pH values: Alter the ionization states of amino acid residues in the active site, disrupting substrate binding and catalysis, leading to denaturation. - **Substrate Concentration:** - Initial increase in activity with increasing substrate concentration (following Michaelis-Menten kinetics). - At very high substrate concentrations, the enzyme becomes saturated, and the reaction rate reaches its maximum ($V_{max}$). - **Enzyme Concentration:** - Reaction rate is directly proportional to enzyme concentration, assuming substrate is not limiting. - **Inhibitors:** Molecules that reduce enzyme activity. - **Reversible:** Competitive, non-competitive, uncompetitive. - **Irreversible:** Covalently bind to the enzyme, permanently inactivating it. - **Activators:** Molecules that increase enzyme activity. ### Mechanism of Enzyme Action Enzymes lower the activation energy of a reaction, thereby increasing its rate, without changing the equilibrium of the reaction. 1. **Binding to Substrate:** The enzyme (E) binds to its specific substrate (S) at a region called the **active site**, forming an enzyme-substrate (ES) complex. * **Lock-and-Key Model:** Old model proposing a rigid active site perfectly complementary to the substrate. * **Induced Fit Model:** Current accepted model, where the active site undergoes a conformational change upon substrate binding, to achieve a more precise fit. 2. **Catalysis:** Within the ES complex, the enzyme facilitates the conversion of substrate to product (P) through various mechanisms: * **Proximity and Orientation:** Bringing substrates together in the correct orientation. * **Strain/Distortion:** Inducing strain in the substrate, making bonds easier to break. * **Acid-Base Catalysis:** Donating or accepting protons. * **Covalent Catalysis:** Forming transient covalent bonds with the substrate. 3. **Product Release:** The enzyme-product (EP) complex dissociates, releasing the product, and the enzyme is regenerated in its original form, ready for another catalytic cycle. $$E + S \rightleftharpoons ES \rightleftharpoons EP \rightleftharpoons E + P$$ ### Coenzymes and Cofactors Many enzymes require non-protein molecules to assist in catalysis. - **Cofactor:** A general term for any non-protein chemical component required for enzyme activity. They can be inorganic ions or complex organic molecules. * **Inorganic Cofactors:** Metal ions like $Zn^{2+}$, $Mg^{2+}$, $Fe^{2+}$, $Cu^{2+}$. They often assist in substrate binding or electron transfer. * **Organic Cofactors:** - **Coenzymes:** Organic molecules that bind loosely or tightly to the enzyme. They often act as transient carriers of specific functional groups (e.g., electrons, atoms). Many are derived from vitamins. * *Examples:* NAD$^+$/NADH (electron transfer), FAD/FADH$_2$ (electron transfer), Coenzyme A (acyl group transfer), ATP (phosphate transfer). - **Prosthetic Groups:** Coenzymes that are very tightly (often covalently) bound to the enzyme. * *Example:* Heme in cytochrome oxidase, biotin in carboxylases. - **Holoenzyme:** The catalytically active enzyme with its bound cofactor(s). - **Apoenzyme:** The inactive protein component of an enzyme, lacking its cofactor(s). $$Apoenzyme + Cofactor \rightleftharpoons Holoenzyme$$