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Enzymes and Coenzymes — Comprehensive Study Notes

Enzymes: Basics and Characteristics

Enzymes are biological catalysts produced by cells; most are proteins and are soluble globular proteins.
Some active enzymes are produced from inactive precursors via proteolytic cleavage. These inactive precursors are called zymogens (e.g., trypsin, pepsin, thrombin).
Enzymes that catalyze the same reaction but differ in structure are termed isozymes (isoenzymes).
Enzymes increase the rate of biochemical reactions by up to $10^{17}$ fold.
They lower the activation energy of a reaction but do not change the equilibrium constant.
Enzyme-catalyzed reactions can be exergonic and spontaneous (can occur without energy input) or endergonic (require energy input and are non-spontaneous).
Energetically favorable (exergonic) reactions (e.g., ATP hydrolysis) are often coupled to unfavorable endergonic reactions in reaction coupling.
The hydrolysis of high-energy phosphoanhydride bonds in ATP releases energy that can power many unfavorable metabolic reactions.
Enzyme-catalyzed reactions are distinguished from simple chemical reactions by their specificity.

Enzyme Action and Energy

Enzymes reduce the activation energy of reactions, allowing faster attainment of the transition state.
When product free energy is lower than substrate free energy, the reaction is exergonic (ΔG < 0) and spontaneous; when higher, it is endergonic (ΔG > 0) and non-spontaneous. At equilibrium, ΔG = 0.
The free energy change of a reaction is ΔG, and the standard free energy change is ΔG° (the value under standard conditions).
Do not confuse ΔG with ΔG° or with ΔG'° (standard physiological conditions at pH = 7). ΔG° and ΔG'° provide a consistent basis for comparing thermodynamic favorability;
ΔG = ΔG° + RT ln Q, where Q is the reaction quotient.
Reaction coupling allows energetically favorable reactions to drive unfavorable ones (e.g., ATP hydrolysis coupled to endergonic processes).
High-energy bonds in certain substrates provide more free energy upon hydrolysis than ATP hydrolysis in some instances (per the example data below).

Gibbs Free Energy Concepts and Standard Conditions

In thermodynamics, G is the energy available to do work at a given temperature and pressure.
Exergonic: $ext{Δ}G < 0$ (energy released); Endergonic: $ext{Δ}G > 0$ (energy absorbed).
At equilibrium: $ext{Δ}G = 0$ .
Relationship to standard free energy: $ext{Δ}G = ext{Δ}G^{\bigcirc} + RT \, ext{ln} Q$ , where Q is the reaction quotient.
Standard conditions provide a basis for comparing thermodynamic favorability; ΔG° is the free energy change under those conditions.
Under standard physiological conditions (pH 7), use ΔG'°; note these standard conditions help compare different reactions.
Reaction coupling and energy flow are central to metabolism; exergonic reactions can drive endergonic steps.

Enzyme Cofactors and Coenzymes

Enzymes may be simple or conjugated proteins.
Apoenzyme: inactive protein lacking cofactors.
Holoenzyme: active enzyme plus cofactor.
Cofactor classes (bold definitions):
- Inorganic metal ions (e.g., Cu^{2+} for lysine oxidase).
- Coenzymes (organic molecules derived from vitamins).
- Prosthetic groups: tightly bound cofactors covalently linked (e.g., heme in cytochromes).
- Cosubstrates: derivatives of B vitamins that must be regenerated (loosely bound to enzyme).
Key terms:
- Prosthetic group: tightly bound to enzyme by covalent interactions.
- Cosubstrates: loosely bound coenzymes that are regenerated after the reaction.
- Holoenzyme: active enzyme with its cofactors.
- Apoenzyme: inactive protein without cofactors.
Zn^2+ as a cofactor: carbonic anhydrase, alkaline phosphatase, DNA polymerase.
Cosubstrates are often vitamin-derived coenzymes that participate in redox or transfer reactions and are regenerated; examples include NAD^+/NADH, NADP^+/NADPH, FAD/FADH2, FMN/FMNH2.
Prosthetic groups are tightly bound cofactors (e.g., heme in cytochromes).
Niacin (Vitamin B3) and Riboflavin (Vitamin B2) derivatives participate as coenzymes in oxidation–reduction reactions:
- NAD^+/NADH and NADP^+/NADPH (niacin-derived).
- FAD and FMN (riboflavin-derived).
The oxidized form of cosubstrates accepts electrons and a proton to become reduced.
Example: Lactate dehydrogenase uses NAD^+ as a coenzyme in lactate/pyruvate conversion.

NAD^+/NADP^+/FAD/FMNs and Vitamin Connections

NAD^+ and NADP^+ are cosubstrates (nicotinamide adenine dinucleotide derivatives).
FAD and FMN are flavin-based coenzymes (riboflavin derivatives).
Niacin (B3) and Riboflavin (B2) are the vitamin precursors for these coenzymes.
The oxidized cosubstrate accepts electrons and protons to become reduced (e.g., NAD^+ → NADH).

Enzyme Nomenclature and EC Classification

Enzymes are classified using an Enzyme Commission (EC) designation, followed by four digits.
The first digit indicates the major class, with subsequent digits narrowing into subcategories.
The six major classes (EC 1–6):
- EC 1: Oxidoreductases
- EC 2: Transferases
- EC 3: Hydrolases
- EC 4: Lyases
- EC 5: Isomerases
- EC 6: Ligases (sometimes called Synthetases)
A more recent addition includes translocases (e.g., Na^+/K^+-ATPase) that move molecules or ions across membranes.
Example: Aminopeptidase has EC number 3.4.11.4, illustrating the hierarchical breakdown:
- EC 3: Hydrolases (act on chemical bonds with water)
- EC 3.4: Hydrolases acting on peptide bonds
- EC 3.4.11: Hydrolases cleaving amino-terminal amino acid from a polypeptide
- EC 3.4.11.4: Those that cleave the amino-terminal end from a tripeptide

Major Enzyme Groups with Examples

Group 1: Oxidoreductases

Function: Transfer of electrons in oxidation–reduction reactions.
Electron donors become oxidized; acceptors become reduced.
Commonly require NAD^+ or FAD as loosely bound coenzymes.
Example: Lactate dehydrogenase (LDH) catalyzes the reversible conversion of lactate to pyruvate with reduction of NAD^+ to NADH + H^+:
$\text{Lactate} + \text{NAD}^+ \rightarrow \text{Pyruvate} + \text{NADH} + \text{H}^+.$
Other example: Alcohol dehydrogenase (as a representative oxidoreductase).
Vitamin context: NAD^+/NADP^+ (niacin) and FAD/FMN (riboflavin) serve as coenzymes.
Often require NAD or FAD as cosubstrates.

Group 2: Transferases

Function: Transfer of functional groups such as carbon, nitrogen, phosphorus, or sulfur groups.
Examples:
- Kinases (e.g., hexokinase in glycolysis; hepatic glucokinase) transfer the γ-phosphoryl group of ATP.
- Transaminases transfer amino groups (–NH_2) in transamination reactions.
Coenzyme for transaminases: Pyridoxal phosphate (PLP), derived from Vitamin B6.
Transamination example (simplified):
- Amino group transfer from an amino acid (e.g., L-Glu) to a keto acid (e.g., pyruvate) to form another amino acid (e.g., L-Ala) and a different keto acid (e.g., α-ketoglutarate).
- General representation: $ext{Amino acid}<em>1 + \text{Keto acid}</em>2 \rightarrow \text{Amino acid}<em>2 + \text{Keto acid}</em>1.$
Hexokinase transfers a phosphate from ATP to glucose (first step of glycolysis):
$\text{Glucose} + \text{ATP} \rightarrow \text{Glucose-6-phosphate} + \text{ADP}.$

Group 3: Hydrolases

Function: Catalyze hydrolysis reactions where H2O is added to break bonds.
General mechanism: A substrate is cleaved into two or more products with incorporation of H and OH from water.
Examples:
- Peptidases/proteases cleave peptide bonds.
- Phosphatases remove phosphate groups.
- Glucosidases degrade glycogen.
Specific example: Glucose + H2O → Glucose-6-phosphate + Pi; glucose-6-phosphatase hydrolyses glucose-6-phosphate, producing free glucose in the liver for diffusion into blood; important in gluconeogenesis (opposes the hexokinase step in glycolysis).
Debranching enzyme (a-1,6-glucosidase) catalyzes hydrolysis of α-1,6-glycosidic bonds in glycogen during glycogenolysis.

Group 4: Lyases

Function: Catalyze cleavage by elimination or addition of a group (often without redox chemistry).
Examples:
- Dehydratases remove H_2O in dehydration reactions.
- Decarboxylases remove CO_2 in decarboxylation reactions (e.g., pyruvate decarboxylase).
- Other examples include fumarase and carbonic anhydrase.
Note: Carbonic anhydrase participates in rapid interconversion of CO2 and water to carbonic acid, protons, and bicarbonate ions in erythrocytes; CO2 travels in blood as bicarbonate and is transported to the lungs for exhalation (via carbonic anhydrase).

Group 5: Isomerases

Function: Transfer or rearrange functional groups within a molecule (isomerization).
Examples:
- Triose phosphate isomerase (glycolysis): interconverts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.
- Epimerase activity (e.g., converting one epimer to another).
- D-Xylulose-5-phosphate and D-Ribulose-5-phosphate are converted via isomerization steps in carbohydrate metabolism.

Group 6: Ligases (Synthetases)

Function: Join two molecules together using energy from ATP hydrolysis; ligation reactions.
Example: Pyruvate carboxylase catalyzes a ligation using ATP energy; important gluconeogenic enzyme. “Ligate” means to bind.

Translocases (newer class)

A recent addition to enzyme classifications includes translocases such as Na^+/K^+ ATPase that move molecules or ions across membranes.

Active Site and Substrate Binding

The active site is the region of the enzyme where catalysis occurs.
Substrate binds to the active site in a cleft on the enzyme; non-covalent interactions (electrostatic, hydrogen bonding, van der Waals) are key.
Lock-and-key model (simple) vs Induced-fit model (recognizes conformational flexibility of the protein to accommodate substrate).
The collection of amino acid side chains in the enzyme’s tertiary structure forms the active site in the correct alignment to bind substrate; residues can be distant in primary sequence but come together in 3D structure.

Enzyme Specificity

Types of specificity:
- Stereochemical specificity: e.g., Lactate dehydrogenase acts on L-lactate; Aspartase acts on L-aspartate.
- Absolute specificity: e.g., Glucokinase; Urease (e.g., Helicobacter pylori context).
- Functional group specificity: e.g., Hexokinase; Alcohol dehydrogenase.
- Bond specificity: catabolic enzymes involved in degradation.
Enzyme specificity is a central concept for understanding enzyme function and metabolism.

Practical Examples and Concepts

Carbonic anhydrase as a physiologically significant enzyme: rapid CO2/H2O ↔ carbonic acid ⇌ bicarbonate/protons in blood; crucial for CO2 transport from tissues to lungs.
ATP and high-energy phosphate bonds: ATP hydrolysis is a canonical high-energy bond reaction; other substrates with high-energy bonds can release more free energy per mole than ATP in certain contexts (per the examples below).
High-energy bond examples (ΔG° values):
- ATP hydrolysis: $\Delta G^{\circ}_{ATP} \approx -7.3 \text{ kcal/mol}$
- 1,3-bisphosphoglycerate: $\Delta G^{\circ} = -11.8 \text{ kcal/mol}$
- Phosphoenolpyruvate (PEP): $\Delta G^{\circ} = -14.8 \text{ kcal/mol}$
- Acetyl-CoA: $\Delta G^{\circ} = -7.7 \text{ kcal/mol}$
These substrates participate in substrate-level phosphorylation steps in glycolysis (e.g., phosphoglycerate kinase, pyruvate kinase).

Poll Prompts and Interactive Slides (from lecture prompts)

Slido prompts included in slides:
- "What term best describes a large inactive enzyme precursor protein that is cleaved to the active enzyme?" (Answer: zymogen).
- "Which enzyme does not require zinc as a cofactor?" (Answer depends on options shown in poll).
- Notes indicate to install Slido app to display poll results on certain slides.

Summary: Key Takeaways

Enzymes increase reaction rates by lowering activation energy without altering equilibrium constants.
They can drive forward and reverse reactions equally by stabilizing the transition state.
Cofactors and coenzymes (often vitamin-derived) are crucial for many enzymatic activities; they include metal ions, prosthetic groups, and cosubstrates.
The active site is the catalysis locus; substrate binding involves noncovalent interactions and can follow lock-and-key or induced-fit models.
Enzymes are classified into six major EC groups: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases; with a newer translocase class added for membrane transport proteins.
EC numbering provides a systematic way to describe enzyme function and specificity (e.g., aminopeptidase EC 3.4.11.4).
Important physiologic enzymes discussed include carbonic anhydrase, lactate dehydrogenase, hexokinase, glucokinase, phosphatases, kinases, and others involved in glycolysis and gluconeogenesis.
The relationship between ΔG, ΔG°, and ΔG'° is essential for understanding thermodynamics in biochemistry; standard conditions enable comparison across reactions and pathways.

Selected Equations and Data (-LaTeX formatted-)

Activation energy concept and rate enhancement (conceptual; no fixed numeric here besides notes):
- Enzyme-catalyzed rate enhancements can reach up to $10^{17}$ fold.
Reaction energetics:
- Exergonic reaction: $\Delta G < 0$ (energy released).
- Endergonic reaction: $\Delta G > 0$ (energy absorbed).
- At equilibrium: $\Delta G = 0$ .
Relationship between free energy changes:
- $\Delta G = \Delta G^{\circ} + RT \ln Q$
- Standard physiologic condition at pH 7: $\Delta G'^{\circ}$ (not to be confused with $\Delta G^{\circ}$ ).
Lactate dehydrogenase reaction:
- $\text{Lactate} + \text{NAD}^+ \rightarrow \text{Pyruvate} + \text{NADH} + \text{H}^+$
Notation for aminopeptidase example:
- EC 3.4.11.4 corresponds to enzymes that cleave the amino-terminal end from a tripeptide.
Table 11-1: Catalytic power of selected enzymes (representative values)
- Carbonic anhydrase: nonenzymatic $1.3\times 10^{-1}\,\text{s}^{-1}$ ; enzymatic $1\times 10^{6}\,\text{s}^{-1}$ ; rate enhancement $7.7\times 10^{6}$ .
- Chorismate mutase: nonenzymatic $2.6\times 10^{-5}\,\text{s}^{-1}$ ; enzymatic $50\,\text{s}^{-1}$ ; rate enhancement $1.9\times 10^{6}$ .
- Triose phosphate isomerase: nonenzymatic $4.3\times 10^{-6}\,\text{s}^{-1}$ ; enzymatic $4300\,\text{s}^{-1}$ ; rate enhancement $1.0\times 10^{9}$ .
- Carboxypeptidase A: nonenzymatic $3.0\times 10^{-9}\,\text{s}^{-1}$ ; enzymatic $578\,\text{s}^{-1}$ ; rate enhancement $1.9\times 10^{11}$ .
- AMP nucleosidase: nonenzymatic $1.0\times 10^{-11}\,\text{s}^{-1}$ ; enzymatic $60\,\text{s}^{-1}$ ; rate enhancement $6.0\times 10^{12}$ .
- Staphylococcal nuclease: nonenzymatic $1.7\times 10^{-1}\,\text{s}^{-1}$ ; enzymatic ??; rate enhancement $5.6\times 10^{14}$ .