Enzymes and Coenzymes — Comprehensive Study Notes

Enzymes and Coenzymes — Comprehensive Study Notes

• Enzymes are biological catalysts produced by cells, most of which are soluble globular proteins.

  • They increase reaction rates by lowering activation energy and do not change the equilibrium constant.

  • They can catalyze exergonic (spontaneous) and endergonic (non-spontaneous, energy-requiring) reactions.

  • Activation energy (Ea) is the energy barrier that must be overcome for a reaction to proceed.

  • Enzyme-catalyzed reactions are distinguished from simple chemical reactions by their high specificity.

• Precursors and related features of enzymes

  • Zymogens: inactive enzyme precursors activated by proteolytic cleavage (examples: trypsin, pepsin, thrombin).

  • Isozymes/isoenzymes: different enzymes that catalyze the same reaction but differ in structure.

  • Enzymes can be activated post-translationally or by proteolysis, enabling regulated activity.

• Energetics and metabolism fundamentals

  • Enzymes lower the activation energy but do not alter the equilibrium constant of a reaction.

  • Reactions can be exergonic (ΔG < 0) or endergonic (ΔG > 0).

  • Reaction coupling: favorable (exergonic) reactions (e.g., ATP hydrolysis) drive unfavorable endergonic reactions by sharing energetic coupling.

  • ATP and high-energy bonds

  • ATP hydrolysis releases energy that can power unfavorable metabolic reactions.

  • Other high-energy phosphate-containing compounds also exist and can release more energy than ATP hydrolysis:

    • 1,3-bisphosphoglycerate: riangle G^{igcirc} = -11.8\ ext{kcal/mol}

    • Phosphoenolpyruvate: riangle G^{igcirc} = -14.8\ ext{kcal/mol}

    • Acetyl-SCoA: riangle G^{igcirc} = -7.7\ ext{kcal/mol}

  • These are key substrate-level phosphorylation steps in glycolysis; phosphoglycerate kinase is involved.

  • Free energy changes in thermodynamics

  • Gibbs free energy, $G$, is the energy available to perform work at a given temperature and pressure.

  • The change in Gibbs free energy for a reaction describes whether a process releases energy (exergonic) or requires energy (endergonic).

  • At equilibrium, $\Delta G = 0$.

  • Standard free energy change, $\Delta G^{\circ}$, and physiological standard free energy change, $\Delta G'^{\circ}$ (with pH 7), provide consistent comparison baselines across reactions.

  • Important distinctions:

    • $\Delta G^{\circ}$ and $\Delta G'^{\circ}$ are standard-state terms; they are not the actual cellular free-energy changes under all conditions.

    • Cellular conditions shift actual free-energy changes, but standard values allow comparison.

  • Reaction coupling examples

  • Hydrolysis of ATP (high-energy phosphoanhydride bonds) powers many unfavorable reactions in metabolism.

• Enzyme cofactors and coenzymes

  • Apoenzyme: inactive protein lacking cofactors.

  • Holoenzyme: active enzyme with its cofactor.

  • Cofactors come in three broad kinds:

  • Inorganic metal ions (e.g., Cu++, Zn2+ as needed by specific enzymes).

  • Coenzymes (organic molecules; often vitamin-derived).

  • Prosthetic groups: tightly bound, often covalently attached; regenerated as part of the reaction sequence (e.g., the heme group in cytochromes).

  • Cosubstrates: derivatives of B vitamins that must be regenerated, loosely bound to the enzyme (e.g., NAD+/NADP+, FAD/FMN).

  • Key vocabulary:

  • Co-factor (general term for non-protein components required for activity).

  • Coenzyme (organic cofactor).

  • Prosthetic group (tightly bound cofactor).

  • Holoenzyme (enzyme + cofactor).

  • Apoenzyme (enzyme without cofactor).

  • Cofactor classification and roles summarized on p 17–18 of the text.

• Specific cofactors and common vitamin-derived cosubstrates

  • NAD+ and NADP+ (niacin or vitamin B3 derivative) serve as electron carriers in oxidation–reduction reactions.

  • Flavin cofactors: FMN and FAD (riboflavin or vitamin B2 derivative) act as cosubstrates in redox reactions.

  • Niacin (B3) and riboflavin (B2) derivatives are common cosubstrates required for redox enzymes.

  • Cosubstrates are often regenerated by other enzymes in the metabolic network.

• Zinc and other metal cofactors

  • Some enzymes require zinc as a cofactor (e.g., carbonic anhydrase, alkaline phosphatase, DNA polymerase).

  • Zinc is an essential metal ion cofactor for cell growth and wound healing.

• Enzyme nomenclature and classification (EC numbers)

  • Enzymes are classified using an EC designation with four digits: EC x.x.x.x.

  • The first digit indicates the major class; subsequent digits specify subcategories.

  • Major classes (the six enzyme categories):

  • Oxidoreductases (Group 1): transfer of electrons in oxidation–reduction reactions; often require NAD+ or FAD as coenzymes.

  • Transferases (Group 2): transfer of functional groups (e.g., carbon, nitrogen, phosphorus, sulfur).

  • Hydrolases (Group 3): catalyze hydrolysis using water; examples include peptidases/proteases, phosphatases, glucosidases.

  • Lyases (Group 4): cleavage by elimination or addition of groups; includes dehydratases and decarboxylases.

  • Isomerases (Group 5): catalyze isomerizations (e.g., epimerases, triose phosphate isomerase).

  • Ligases (Group 6): join two molecules (often using ATP) to form bonds; e.g., pyruvate carboxylase (a ligase).

  • A newer class, translocases (e.g., Na+/K+ ATPase), moves molecules or ions across membranes.

  • Example: aminopeptidase has EC 3.4.11.4, illustrating how EC digits map to enzyme subcategories:

  • EC 3: hydrolases

  • EC 3.4: hydrolases acting on peptide bonds

  • EC 3.4.11: amino-terminal aminopeptidases

  • EC 3.4.11.4: cleave amino-terminal end from a tripeptide

• Enzyme–substrate interaction and active site

  • Substrate binds to the active site, a cleft on the enzyme, via noncovalent interactions (key for binding affinity and orientation).

  • The active site is the region where catalysis occurs; precise alignment of catalytic residues enables reaction chemistry.

  • The arrangement of amino acid side chains in the enzyme’s tertiary structure forms the active site, which can bring substrates into close proximity and stabilize transition states.

  • The concept of an active site can place substrates in a specific geometry required for the reaction to proceed.

• Enzyme specificity and types of specificity

  • Enzyme specificity refers to the enzyme’s preference for substrates and reaction type.

  • Types of specificity described:

  • Stereochemical specificity: e.g., lactate dehydrogenase acts on L-lactate; aspartase acts on L-aspartate.

  • Absolute specificity: e.g., glucokinase acts only on glucose; urease (e.g., in Helicobacter pylori) acts on a specific substrate.

  • Functional group specificity: e.g., hexokinase acts on glucose’s functional groups; alcohol dehydrogenase on alcohol substrates.

  • Bond specificity: enzymes catalyzing specific bond cleavages relevant to catabolic pathways.

  • Visual aids note that enzyme specificity is a central concept in enzyme kinetics and metabolism.

• Models of substrate binding

  • Lock-and-key model (classic view): substrate fits an enzyme’s active site as a perfect match.

  • Induced-fit model (Koshland): enzyme undergoes conformational changes to better fit the substrate, accounting for protein flexibility.

  • The active site is formed by a collection of amino acid side chains that come together in the enzyme’s tertiary structure to bind the substrate; residues can be distant in the primary sequence but come into proximity in 3D space.

• The active site and enzyme–substrate interaction (concept recap)

  • Catalysis occurs within the active site by aligning substrates and catalytic residues, often using noncovalent interactions to orient and stabilize the transition state.

  • The active site provides a microenvironment that can modulate pK values and reaction pathways.

• Enzyme action and Gibbs free energy changes

  • Enzymes reduce the activation energy of reactions, making them faster, without changing the overall free energy change (ΔG) of the reaction.

  • If products have lower free energy than substrates, the reaction is exergonic (ΔG < 0); if higher, endergonic (ΔG > 0).

  • At equilibrium, ΔG = 0. In enzymatic terms, the enzyme stabilizes the transition state to accelerate both forward and reverse reactions equally.

  • Distinguish between ΔG (actual cellular context) and ΔG° (standard free energy change under standard conditions) and ΔG′° (physiological standard conditions, pH 7).

  • These standard values provide a consistent basis for comparing thermodynamic favorability across reactions.

  • See p 16–17 discussion in the text for deeper thermodynamics context.

• Coupled reactions and high-energy bonds in metabolism

  • Exergonic reactions can drive endergonic reactions via coupling, often through shared intermediates or energy transfer.

  • High-energy bonds in metabolites yield large negative ΔG° values upon hydrolysis, enabling energy transfer to other processes.

  • ATP is the prototypical example, but other compounds with high-energy bonds include:

  • 1,3-bisphosphoglycerate: $\triangle G^{\circ} = -11.8\ \,\text{kcal/mol}$

  • Phosphoenolpyruvate: $\triangle G^{\circ} = -14.8\ \,\text{kcal/mol}$

  • Acetyl-CoA: $\triangle G^{\circ} = -7.7\ \,\text{kcal/mol}$

  • These components participate in substrate-level phosphorylation steps in glycolysis (e.g., phosphoglycerate kinase step).

• Substrate turnover rates and catalytic power (Table 11-1 reference)

  • Enzymes can accelerate reactions by many orders of magnitude relative to the nonenzymatic rate.

  • Example values (nonenzymatic rate, enzymatic rate, rate enhancement):

  • 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 $4.3\times 10^{3}\ \text{s}^{-1}$; rate enhancement $1.0\times 10^{9}$.

  • Carboxypeptidase A: nonenzymatic $3.0\times 10^{-9}\ \text{s}^{-1}$; enzymatic $5.78\times 10^{2}\ \text{s}^{-1}$; 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 ??? (formatting in table shows garbled value); rate enhancement $5.6\times 10^{14}$.

  • Source: Radzicka, A. and Wolfenden, R., Science 267, 91 (1995).

• Enzyme–substrate interaction visuals (collective concepts from figures in the course text)

  • Substrate binds to the active site in a cleft on the enzyme; noncovalent interactions govern binding.

  • Lock-and-key vs induced-fit models illustrate how enzyme conformational flexibility accommodates substrates and facilitates catalysis.

  • The active site is formed by amino acid side chains brought together in the enzyme’s tertiary structure; residues may be distant in the primary sequence but align in 3D space to enable binding and catalysis.

• Oxidoreductases (Group 1)

  • Transfer of electrons in oxidation–reduction reactions.

  • Typical electron donors/acceptors: NAD+ (niacin/B3) or FAD (riboflavin/B2) as loosely bound coenzymes.

  • Example: lactate dehydrogenase (LDH) catalyzes the reversible conversion of lactate to pyruvate while reducing NAD+ to NADH + H+.

  • Reaction example: $\text{Lactate} + \mathrm{NAD}^+ \rightleftharpoons \text{Pyruvate} + \mathrm{NADH} + \mathrm{H}^+.$

• Lactate dehydrogenase (LDH) specific overview

  • Function: catalyzes reversible conversion of lactate to pyruvate with NAD+/NADH cycling.

  • NAD+/NADH roles as electron carriers in redox chemistry.

  • Structural and catalytic details reinforce LDH as a prototype oxidoreductase.

• Transferases (Group 2) and key examples

  • Transfer functional groups (C, N, P, S) between molecules.

  • Kinases (e.g., hexokinase, hepatic glucokinase) transfer the γ-phosphoryl group from ATP.

  • Transaminases transfer amino groups (–NH2) in transamination reactions.

  • Coenzyme for transaminases: Pyridoxal phosphate (PLP) derived from vitamin B6.

• Transamination example (illustrated reaction)

  • Amino group transfer from an amino acid to a keto acid acceptor (example given with glutamic acid and alanine).

  • General depiction:

  • Amino group transfer from amino acid₁ (e.g., L-glutamic acid) to keto acid₂ (e.g., pyruvic acid) yielding amino acid₂ (e.g., L-alanine) and a corresponding keto acid (e.g., α-ketoglutaric acid).

• Hexokinase (Group 2 example)

  • Hexokinase transfers a phosphate group from ATP to glucose, forming glucose-6-phosphate.

  • This is the first committed step in glycolysis.

• Hydrolases (Group 3)

  • Catalyze hydrolysis reactions where water is added to break bonds.

  • Examples include:

  • Peptidases/proteases cleaving peptide bonds.

  • Phosphatases removing phosphate groups.

  • Glucosidases degrading glycogen.

  • Example hydrolysis: Glucose-6-phosphate + Pi from Glucose-6-phosphatase yields free glucose (liver) enabling diffusion into bloodstream for usage by brain and skeletal muscle; important in gluconeogenesis (opposes the hexokinase step).

  • Debranching enzyme (a-1,6-glucosidase) catalyzes hydrolysis of α-1,6-glycosidic bonds in glycogen during glycogenolysis.

• Lyases (Group 4)

  • Catalyze cleavage by elimination or addition of a group.

  • Subtypes include dehydratases (remove H2O) and decarboxylases (remove CO2).

  • Examples include fumarase and carbonic anhydrase.

• Carbonic anhydrase (Group 4, lyase example)

  • In erythrocytes, rapidly interconverts CO2 and water to carbonic acid, protons, and bicarbonate ions.

  • CO2 can travel in blood as bicarbonate; carbonic anhydrase converts bicarbonate back to CO2 in lungs for exhalation.

• Pyruvate decarboxylase (Group 4, lyase example)

  • Participates in yeast fermentation; distinct from pyruvate dehydrogenase (an oxidoreductase).

• Isomerases (Group 5)

  • Transfer groups within molecules to create isomers.

  • Example: Triose phosphate isomerase in glycolysis; interconverts glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

  • Epimerase and other isomerases also fall into this group.

• Pyruvate carboxylase (Group 6) and ligases

  • Pyruvate carboxylase catalyzes a ligation (joining) reaction using ATP energy; important in gluconeogenesis.

  • Ligases (synthetases) form bonds by consuming ATP.

• Translocases (newest class mentioned)

  • Enzymes that move molecules or ions across membranes (e.g., Na+/K+ ATPase).

• Summary points to emphasize

  • Enzymes increase reaction rates by lowering activation energy without altering equilibrium.

  • They accelerate forward and reverse reactions similarly by stabilizing the transition state.

  • Some enzymes require metal ions or organic prosthetic groups/coenzymes (often B vitamin derivatives).

  • The active site is the catalytic center of the enzyme.

  • Enzyme classification uses EC numbers (four digits) to denote major class, subclass, sub-subclass, and individual enzyme.

  • The six major enzyme classes are oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

• Quick practice prompts (as in the lecture/polls)

  • What term best describes a large inactive enzyme precursor that is cleaved to become active? (Answer: zymogen)

  • Which enzyme does not require zinc as a cofactor? (Refer to in-class poll questions.)

  • What type of enzyme does lactate dehydrogenase belong to? (Answer: Oxidoreductase)

• Connections to broader biochemistry foundations

  • The concepts of activation energy, energy coupling, and the role of cofactors link directly to metabolic control, flux through pathways, and energy economy in cells.

  • The EC-number system provides a standardized language for enzyme identification across disciplines and organisms.

• Practical and ethical considerations

  • Understanding enzyme inhibitors and activators has implications for drug design and disease treatment.

  • Knowledge of cofactor requirements informs nutritional guidelines (e.g., niacin and riboflavin intake affecting redox enzyme activity).

• Notable formulas and numerical references

  • Oxidoreductase reaction example (LDH):

  • $\text{Lactate} + \mathrm{NAD}^+ \rightleftharpoons \text{Pyruvate} + \mathrm{NADH} + \mathrm{H}^+.$

  • Gibbs free energy concepts: $\Delta G$, $\Delta G^{\circ}$, and $\Delta G'^{\circ}$ with the understanding that standard values are used for comparison but actual cellular values may differ.

  • High-energy bonds in metabolism (examples):

  • $\Delta G^{\circ}_{ATP\text{ hydrolysis}} \approx -7.3\ \text{kcal/mol}$

  • $\Delta G^{\circ}$ for 1,3-bisphosphoglycerate = $-11.8\ \text{kcal/mol}$

  • $\Delta G^{\circ}$ for phosphoenolpyruvate = $-14.8\ \text{kcal/mol}$

  • $\Delta G^{\circ}$ for acetyl-CoA = $-7.7\ \text{kcal/mol}$

• References to figures and text sections mentioned in the transcript

  • Substrate binding: Figure 3.1, page 15 text – enzyme–substrate complex via active-site cleft and noncovalent interactions.

  • Section references: discussion of Gibbs free energy and standard states (pp. 15–17); enzyme cofactors and classification (pp. 16–19).

  • Table 11-1: Catalytic power of several enzymes (Rate enhancements up to ~10^14–10^15 for some enzymes).

  • Carbonic anhydrase discussion and physiologic role (pp. 19–20 and Fig. 3.4).

• Key terms to memorize

  • Apoenzyme, Holoenzyme, Coenzyme, Cofactor, Prosthetic group, Cosubstrate, Isoenzyme, Zymogen, Active site, Lock-and-key, Induced fit, EC numbers, Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases, Translocases

• Quick glance: practical examples to remember

  • LDH: oxidoreductase; lactate ↔ pyruvate with NAD+/NADH cycling.

  • Hexokinase: transferase; first step of glycolysis; ATP-dependent phosphorylation of glucose.

  • Carbonic anhydrase: lyase; rapid CO2–H2O → carbonic acid equilibrium; critical in CO2 transport in blood.

  • Pyruvate carboxylase: ligase; ATP-dependent carboxylation in gluconeogenesis.

  • Debranching enzyme (a-1,6-glucosidase): hydrolase; glycogen breakdown.

• Final takeaway

  • Enzymes are central to controlling metabolism due to their specificity, regulation via cofactors and zymogen activation, and the ability to couple energetically favorable and unfavorable reactions to drive essential cellular processes.