Enzyme Catalysis and Regulation of Enzyme Activity Study Guide

General Properties of Enzymes as Nature’s Catalysts

• Definition and Composition:     * Nearly all known enzymes are proteins. There are exceptions to this rule (though the specific non-protein exceptions were not named in this context).     * Enzymes act as catalysts, which are substances that increase the rate of a chemical reaction without being consumed by the reaction.

• Two Remarkable Features of Enzymes:     1. Catalytic Power: Enzymes are capable of speeding up reactions, usually by more than $10^6$ fold compared to uncatalyzed reactions.     2. Specificity: Enzymes usually catalyze only one specific reaction or a small family of closely related reactions.         * Example (Trypsin): This enzyme specifically hydrolyzes peptide bonds on the carboxyl side of the amino acids Lysine or Arginine.         * Example (Aspartase): This enzyme catalyzes the reaction: $H^+ + ext{aspartate} \rightarrow ext{fumarate} + NH_4^+$. It is highly stereospecific, recognizing only the L-stereoisomer of aspartate.

Rate Enhancements Produced by Enzymes

• Quantitative Enhancements (Table 6-5):     * Cyclophilin: $10^5$     * Carbonic anhydrase: $10^7$     * Triose phosphate isomerase: $10^9$     * Carboxypeptidase A: $10^{11}$     * Phosphoglucomutase: $10^{12}$     * Succinyl-CoA transferase: $10^{13}$     * Urease: $10^{14}$     * Orotidine monophosphate decarboxylase: $10^{17}$

Classification of Enzymes

• IUB Classification System: Enzymes are classified into six major classes based on the type of reaction they catalyze.

• The Six Major Classes:     1. Oxidoreductases: Catalyze redox (oxidation-reduction) reactions involving the transfer of electrons. Examples include Oxidases, Reductases, and Dehydrogenases (e.g., Alcohol dehydrogenase).     2. Transferases: Catalyze the transfer of functional groups (such as methyl, acyl, or phosphate groups) from one molecule to another. Examples include Transaminase, Transketolase, Transaldolase, and Kinases (e.g., Glukokinase or Hexokinase).     3. Hydrolases: Catalyze the breakage of bonds through the addition of water ($H_2O$). Examples include Amylases, Lipases, Proteases, and Nucleases (e.g., Sucrase).     4. Lyases (or Desmolases): Catalyze the breakage of bonds (often forming double bonds or removing groups) without the use of water or redox reactions. Examples include Aldolase, Fumarase, and Decarboxylases (e.g., Histidine decarboxylase).     5. Isomerases: Catalyze the rearrangement of groups within a single molecule to produce structural or geometric isomers. Examples include Isomerases, Mutases, and Epimerases (e.g., Glucose-6-Phosphate Isomerase).     6. Ligases (or Synthetases): Catalyze the binding of two molecules or groups together, a process usually coupled with the hydrolysis of $ATP$. Examples include Synthetases and Carboxylases (e.g., Pyruvate carboxylase).

• Enzyme Commission (EC) Numbers:     * Enzymes are assigned a four-digit EC classification number.     * First Digit: Represents the major class (1 through 6).     * Second and Third Digits: Represent the substrate subclass and sub-subclasses.     * Final Digit: A serial number assigned to that specific enzyme.

Isozymes (Isoenzymes)

• Definition: Numerous examples of enzymes that catalyze the very same chemical reaction but differ in their primary protein structure and usually in their kinetic properties.

• Functional Significance:     * Isozymes are often restricted to specific tissue types, organelles, or developmental periods.     * Tissue-specific isozymes allow for the diagnosis of specific organ damage. If these enzymes are identified in the blood (outside their native tissues), it indicates cellular leakage or damage.

• Lactate Dehydrogenase (LDH) - A Clinical Example:     * Structure: LDH has a quaternary structure; it is a tetramer consisting of two different types of subunits: H (Heart) and M (Muscle).     * Five Possible Isozymes:         1. $LDH_1 = H_4$         2. $LDH_2 = H_3M$         3. $LDH_3 = H_2M_2$         4. $LDH_4 = HM_3$         5. $LDH_5 = M_4$     * Distribution: Heart LDH consists primarily of H subunits, while liver and skeletal muscle LDH contains M subunits.     * Identification via Electrophoresis: Although subunits have similar molecular weights, their net negative charges differ in the order: H_4 > H_3M > H_2M_2 > HM_3 > M_4. When placed in a medium (like cellulose acetate) and subjected to an electrical current, they migrate at different rates toward the positive electrode.     * Clinical Note: While LDH profiles have historically been diagnostic markers for various pathological states, myocardial infarction is now primarily diagnosed through troponin levels.

Enzyme Cofactors

• Definitions:     * Cofactor: A non-protein component required by some enzymes for biological activity.     * Apoenzyme: The purely protein component of an enzyme that requires a cofactor but does not currently have one bound.

• Types of Cofactors:     1. Coenzymes: These are often derived from water-soluble vitamins. They participate in reactions such as redox reactions, acyl transfer, and carboxylation.     2. Metal Ions: Usually held by coordinate binding with amino acid side chains or a prosthetic group (e.g., haem).

Mechanism of Enzyme Catalysis

• How Enzymes Work:     * Enzymes increase reaction rates by influencing bond stabilities and/or providing an alternate reaction pathway.     * The primary result is the lowering of the Activation Energy ($\Delta G^\ddagger$).     * Reaction steps: Substrate binds to enzyme to form the Enzyme-Substrate (ES) complex, which is then converted into the Transition State Complex (TSC).

• The Transition State Complex (TSC):     * The TSC has the highest free energy in the reaction pathway and is the rarest species.     * The TSC readily converts to products.     * Crucially, the transition state has a lower free energy in the presence of an enzyme compared to an uncatalyzed reaction.

• Thermodynamic Boundaries:     * Enzymes enable equilibria to be reached more quickly.     * Enzymes DO NOT alter the equilibrium constant ($K_{eq}$) or the standard free energy change ($\Delta G^0$) of a reaction.     * Forward and reverse reactions are increased by the same amount.

Substrate Binding and Active Sites

• Binding Characteristics:     * Binding determines enzyme specificity.     * Substrates usually bind via non-covalent interactions: Ionic interactions or Hydrogen bonds. Covalent binding is rare for initial substrate attachment.     * Some enzymes bind more than one substrate, often with different affinities.     * Multi-point binding occurs at the active site.

• Active Site Structure:     * Can be a crevice (e.g., Papain and Chymotrypsin) or a deep pit (e.g., Carbonic anhydrase).     * The shape of the pit or crevice must allow substrate access.     * Amino acids forming the active site often come from distant parts of the linear peptide chain but are brought together by the 3D folding of the protein.     * Example (Pancreatic ribonuclease): A chain of 124 amino acids where $His_{12}$ and $His_{119}$ are both part of the active site.

• Specificity in Serine Proteases:     * Chymotrypsin: Binds aromatic amino acids: Phe, Tyr, Trp.     * Trypsin: Binds basic amino acids: Arg, Lys.     * Elastase: Binds small, non-polar amino acids: Gly, Ala.

Models of Substrate Binding

• Lock and Key Model: Suggests the enzyme and substrate have complementary rigid shapes. While useful, it is considered too simplistic.
• Induced Fit Model: A more accurate model where the binding of the substrate induces a conformational change in the enzyme, leading to a better fit. (Example: Hexokinase).
• The Problem with 'Lock and Key': If an enzyme were perfectly complementary to the substrate, it would stabilize the substrate and increase the activation energy required to reaching the transition state, effectively slowing the reaction. For catalysis to occur, the enzyme must be complementary to the transition state of the reaction, not the substrate itself.

Case Study: Chymotrypsin Mechanism

• Classification: A protease and a serine protease.

• Catalytic Triad: Three specific amino acids at the active site: ASP-HIS-SER ($Asp_{102}$, $His_{57}$, $Ser_{195}$).

• Mechanism Phases:     1. Acylation Phase:         * Active site binding occurs.         * $Asp_{102}$ increases the $pKa$ of $His_{57}$, which then attracts a proton from $Ser_{195}$.         * The "agitated" oxygen in $Ser_{195}$ attacks the carbon in the peptide substrate.         * An unstable tetrahedral intermediate (transition state) is formed.         * The peptide bond breaks. $His_{57}$ donates a proton to the newly formed amino end of the polypeptide, which is then released.         * The carboxyl-terminal peptide remains covalently bound to the enzyme.     2. Deacylation Phase:         * A water molecule splits; the hydroxyl group attacks the carbon of the substrate (covalently bound to the enzyme).         * $His_{57}$ picks up a proton.         * A second unstable tetrahedral intermediate is formed.         * The carbon forms a double bond with oxygen, causing the release of the carboxyl-terminal protein from $Ser_{195}$.         * The $Ser_{195}$ oxygen reacts with the $His_{57}$ proton to restore the active site to its original state.

Regulation of Enzyme Activity

• The Necessity of Regulation: Enzymes simply perform chemistry if substrate is available and thermodynamics are favorable. Regulation is required to ensure cells only generate/store energy or synthesize/break down compounds under appropriate conditions.

• Mechanisms of Control:     1. Genetic Control: Regulation of the transcription of the gene encoding the enzyme.     2. Zymogen Activation: Activating an inactive enzyme precursor through proteolysis.     3. Covalent Modification: The making or breaking of covalent bonds, most notably through phosphorylation.     4. Allosteric Regulation: Binding a modulator to a site other than the active site.

• Allosteric Enzymes:     * "Allosteric" means "another shape."     * Regulated positively or negatively by a modulator/regulator binding to a site distant from the active site.     * They do not display normal Michaelis-Menten enzyme kinetics.     * Usually involved in multi-enzyme pathways, typically catalyzing the first step.     * The regulator is often the end product of the pathway (feedback inhibition).     * Often consist of multi-subunit proteins. Modulator binding on one subunit changes the conformation of the whole protein, altering substrate affinity at the active site on a different subunit.

• Covalent Modulation Examples:     * Phosphorylation: Regulated by kinases (add phosphate) and phosphatases (remove phosphate).         * Glycogen Phosphorylase: Catalyzes: $\text{Glycogen}<em>n + ext{phosphate} \rightarrow ext{glucose-1-phosphate} + \text{Glycogen}</em>{n-1}$.         * Glycogen phosphorylase a: The phosphorylated, active form.         * Glycogen phosphorylase b: The non-phosphorylated, less active form.         * Conversion to 'a' form is catalyzed by Phosphorylase Kinase (2ATP ightarrow 2ADP).         * Conversion to 'b' form is catalyzed by Phosphorylase Phosphatase (2H_2O ightarrow 2P_i).     * Zymogens: Inactive proteins activated by the hydrolysis of specific peptide bonds.         * Trypsinogen: Activated to Trypsin by enterokinase.         * Chymotrypsinogen: Activated to Chymotrypsin by trypsin and self-digestion.         * Pepsinogen: Activated to Pepsin through autolytic activation or $H^+$ (acidic) conditions.         * This protects the producing cell from the proteolytic activity of the enzyme.