Unit 5: Enzymes, Vitamins and Minerals

I. Orientation — Biological catalysis and micronutrient function

Enzymes are biological catalysts that accelerate chemical reactions by lowering activation energy without being permanently consumed. Vitamins and minerals support metabolism mainly as coenzymes, cofactor components, structural elements, or regulators rather than as energy-yielding nutrients themselves.

• Defining properties: Enzymes are usually proteins; catalytic RNA molecules called ribozymes are important exceptions.
• Governing principle: Enzyme activity depends on the formation and conversion of an enzyme–substrate complex, written as (E+S \rightleftharpoons ES \rightarrow E+P).
• Specificity: An enzyme recognizes particular substrates because the active site has complementary shape, charge, and chemical groups.
• Reaction equilibrium: Enzymes increase the rate at which equilibrium is reached but do not change the reaction’s equilibrium constant or overall free-energy change.
• Micronutrient convention: Vitamins are organic compounds required in small amounts; minerals are inorganic elements required in ionic or elemental form.
• Nutritional distinction: Essential nutrients cannot be synthesized in sufficient quantities by the body and must be obtained from the diet.

II. Mechanisms of Enzyme Action — Active sites and transition states

A. Mechanisms of enzymes action

Enzyme action depends on selective substrate binding followed by chemical stabilization of the transition state, the high-energy arrangement between reactants and products.

• Active site: The active site is a three-dimensional pocket containing binding residues and catalytic residues; for example, serine proteases use serine, histidine, and aspartate in catalysis.
• Induced-fit model: Substrate binding can alter enzyme shape so catalytic groups align correctly; hexokinase closes around glucose and ATP after binding.
• Transition-state stabilization: Enzymes bind the transition state more strongly than the ground-state substrate, reducing the activation-energy barrier (E_a).
• Catalytic strategies:
  • Acid–base catalysis: Amino-acid side chains donate or accept protons; histidine commonly performs both roles.
  • Covalent catalysis: A temporary covalent enzyme–substrate bond forms, as in serine proteases.
  • Metal-ion catalysis: Metals such as (Zn^{2+}), (Mg^{2+}), or (Fe^{2+}) stabilize charges or activate water.
  • Proximity and orientation: Binding increases effective local concentration and positions reacting groups precisely.
• Cofactor dependence: An inactive apoenzyme becomes an active holoenzyme after association with its required cofactor, such as (Zn^{2+}) in carbonic anhydrase.
• Reversibility: The same enzyme can catalyze forward or reverse reactions, depending on substrate and product concentrations.

III. Nomenclature and Classification of Enzymes — Naming catalytic function

A. Nomenclature and classification of enzymes

Enzyme names identify the substrate or reaction and are standardized by the Enzyme Commission (EC) system, which assigns four-part numerical classifications.

• Common names: Names often end in “-ase” and indicate the substrate or function, such as lactase, urease, or DNA polymerase.
• Systematic names: A systematic name describes both substrate and reaction; lactate dehydrogenase is formally an oxidoreductase acting on lactate.
• EC numbering: The first number identifies the major reaction class, followed by subclass, sub-subclass, and serial number.
• Major classes:
  • Oxidoreductases (EC 1): Catalyze oxidation–reduction reactions; dehydrogenases transfer hydrogen or electrons.
  • Transferases (EC 2): Transfer functional groups; kinases transfer phosphate from ATP.
  • Hydrolases (EC 3): Break bonds using water; proteases hydrolyze peptide bonds.
  • Lyases (EC 4): Add or remove groups without hydrolysis or oxidation, often forming double bonds.
  • Isomerases (EC 5): Rearrange atoms within a molecule; phosphoglucose isomerase converts glucose-6-phosphate to fructose-6-phosphate.
  • Ligases (EC 6): Join molecules using energy, commonly from ATP; DNA ligase seals DNA breaks.
  • Translocases (EC 7): Move ions or molecules across membranes, often coupled to ATP hydrolysis.
• Functional distinction: “Kinase” generally transfers phosphate to a substrate, whereas “phosphatase” removes phosphate by hydrolysis.

IV. Factors Affecting Enzyme Activity — Conditions controlling rate

A. Factors affecting enzyme activity

Enzyme reaction rate changes when conditions alter molecular collisions, active-site structure, substrate availability, or enzyme concentration.

• Temperature: Rate usually rises as temperature increases because collisions become more frequent; human enzymes often function near (37^\circ\text{C}), while excessive heat denatures proteins.
• pH: Each enzyme has an optimum pH that preserves active-site ionization; pepsin works near pH 2, whereas many cytosolic enzymes function near pH 7.
• Substrate concentration: At low substrate concentration, rate rises approximately in proportion to ([S]); at high concentration, active sites become saturated.
• Enzyme concentration: With substrate in excess, initial rate is proportional to total enzyme concentration. Doubling enzyme concentration approximately doubles (V_0).
• Product concentration: Accumulated product may slow a reaction through product inhibition or by shifting a reversible reaction toward reactants.
• Inhibitors and activators: Heavy metals can inhibit by binding sulfhydryl groups, while required metal ions or allosteric activators can increase activity.
• Ionic strength: Salt concentration affects electrostatic interactions and protein solubility; extreme ionic strength can disrupt enzyme structure.
• Compartmentalization: Cellular location controls exposure to substrates and regulators; lysosomal enzymes, for example, operate in acidic lysosomes.

V. Enzyme Kinetics — Quantifying catalytic rate

A. Enzyme kinetics

Enzyme kinetics measures reaction velocity under defined conditions and provides parameters describing affinity and catalytic capacity.

• Initial velocity: (V_0) is measured before substantial substrate depletion or product accumulation, making the rate approximately constant.
• Michaelis–Menten model: For a simple one-substrate reaction,

V0 = (Vmax [S]) / (Km + [S])

Here, (V0) is initial velocity, (V{\max}) is the maximum velocity at saturation, ([S]) is substrate concentration, and (K_m) is the substrate concentration at which (V0=\frac{1}{2}V{\max}).

• Meaning of (K_m): A lower (K_m) generally indicates that half-maximal velocity is reached at lower substrate concentration; it is often used as an apparent measure of substrate affinity.
• Meaning of (V_{\max}): (V_{\max}) depends on enzyme concentration and catalytic turnover. The turnover number is

kcat = Vmax / [E]total

where (k{\text{cat}}) is turnover number in (\text{s}^{-1}), and ([E]{\text{total}}) is total enzyme concentration.

• Worked example: If (V_{\max}=100\ \mu\text{mol min}^{-1}), (K_m=2\ \text{mM}), and ([S]=2\ \text{mM}), then (V_0=50\ \mu\text{mol min}^{-1}), because ([S]=K_m).
• Saturation curve: A plot of (V_0) against ([S]) is hyperbolic for many Michaelis–Menten enzymes.
• Allosteric enzymes: Multisubunit regulatory enzymes often produce a sigmoidal velocity curve because substrate binding at one site affects other sites; hemoglobin is an analogous cooperative, though nonenzymatic, protein.
• Rate conditions: Kinetic constants are meaningful only when pH, temperature, ionic strength, substrate identity, and enzyme preparation are controlled.

VI. Overview of enzyme inhibition — Regulation and loss of activity

A. Overview of enzyme inhibition

Enzyme inhibition decreases catalytic activity and may be reversible through noncovalent binding or irreversible through permanent chemical modification.

• Reversible inhibition: Inhibitor binding is governed by equilibria and can often be reduced by dilution or removal of inhibitor.
• Competitive inhibition: Inhibitor competes with substrate for the active site. Increasing ([S]) can overcome it; apparent (Km) increases, while (V{\max}) remains unchanged.
• Noncompetitive inhibition: A pure noncompetitive inhibitor binds enzyme and enzyme–substrate complex equally well. (V_{\max}) decreases, while (K_m) is unchanged.
• Uncompetitive inhibition: Inhibitor binds only the enzyme–substrate complex. Both apparent (Km) and (V{\max}) decrease.
• Mixed inhibition: Inhibitor binds free enzyme and enzyme–substrate complex with different affinities; (V_{\max}) decreases and (K_m) may increase or decrease.
• Irreversible inhibition: Covalent or extremely tight binding permanently removes active enzyme; organophosphates inhibit acetylcholinesterase by modifying an active-site serine.
• Allosteric inhibition: A regulator binds a site distinct from the active site and changes conformation; feedback inhibition commonly controls the first committed step of a metabolic pathway.
• Physiological significance: Inhibition permits pathway regulation, protects cells from excessive product formation, and provides drug targets such as cyclooxygenase inhibition by aspirin.

VII. Introduction to vitamins and minerals — Essential micronutrients

A. Introduction to vitamins and minerals

Vitamins and minerals are required in small quantities but support energy metabolism, antioxidant defense, gene regulation, fluid balance, and skeletal or tissue structure.

• Vitamins: Vitamins are organic micronutrients classified by solubility:
  • Fat-soluble: Vitamins A, D, E, and K are stored in liver or adipose tissue; excessive intake can cause toxicity.
  • Water-soluble: Vitamin C and the B-group vitamins are generally less extensively stored and require regular intake, although vitamin B(_{12}) is stored substantially in the liver.
• Coenzyme function: Several vitamins become coenzymes; niacin forms NAD(^+)/NADP(^+), riboflavin forms FAD/FMN, and thiamine forms thiamine pyrophosphate.
• Vitamin examples:
  • Vitamin A: Retinal supports vision; deficiency can cause night blindness.
  • Vitamin D: Promotes calcium and phosphate homeostasis and supports bone mineralization.
  • Vitamin C: Required for collagen hydroxylation and enhances non-heme iron absorption.
  • Vitamin K: Required for activation of several blood-clotting proteins through (\gamma)-carboxylation.
• Minerals: Minerals are inorganic elements divided by required quantity:
  • Major minerals: Calcium, phosphorus, magnesium, sodium, potassium, chloride, and sulfur are needed in relatively larger amounts.
  • Trace elements: Iron, zinc, copper, iodine, selenium, manganese, molybdenum, and chromium are required in smaller amounts.
• Mineral functions:
  • Iron: Forms part of hemoglobin and cytochromes; deficiency impairs oxygen transport.
  • Iodine: Required for thyroid hormones (T_3) and (T_4).
  • Calcium and phosphorus: Provide hydroxyapatite structure in bone and participate in signaling and phosphorylation.
  • Sodium and potassium: Establish electrochemical gradients used in nerve impulses and membrane transport.
  • Zinc and selenium: Zinc supports numerous enzymes and zinc-finger proteins; selenium forms part of glutathione peroxidases.
• Absorption and balance: Nutrient status depends on intake, intestinal absorption, storage, transport, metabolism, and excretion; vitamin D increases intestinal calcium absorption, illustrating coordinated regulation.
• Deficiency and excess: Deficiency disrupts specific biochemical pathways, while excess may be toxic—for example, iron overload damages tissues and excessive vitamin A affects liver and bone.