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Enzymes: Properties & Factors Affecting Activity

For PharmD 1st Year Students

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PART A — PROPERTIES OF ENZYMES

1. Enzymes Are Proteins and Act as Biological Catalysts

"Enzymes do not affect the value of the equilibrium constant between reactants and products. In a reversible reaction, they accelerate forward and reverse reactions by the same relative amount." — Henry's Clinical Diagnosis and Management by Laboratory Methods

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2. Enzyme Specificity

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3. Active Site & Models of Binding

• Lock-and-Key Model: The active site is pre-formed and complementary in shape and charge to the substrate. No conformational change occurs upon binding.
• Induced Fit Model: Substrate binding causes a conformational change in the enzyme, improving the "fit" and lowering activation energy. This is the more widely accepted model.

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4. Enzyme-Substrate (ES) Complex

1. Recognition step: Enzyme binds substrate via ionic interactions, hydrophobic interactions, hydrogen bonds, and van der Waals forces
2. Catalytic step: ES complex decomposes to yield product (P) and free enzyme (E), which is then available for another reaction cycle

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5. Enzyme Concentration and Regulation

• Enzyme levels depend on rate of synthesis (transcriptional/translational control) and rate of degradation
• Substrate presence or inducing molecules can cause a rapid increase in enzyme levels
• Enzymatic activity is regulated by small molecule binding (allosteric regulation) — altering substrate affinity or catalytic activity
• In eukaryotes, different organs express different isoforms (isozymes) of the same enzyme

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6. Types of Catalytic Mechanisms

• Acid-base catalysis — histidine side chain (pKa ~6 makes it ideal)
• Covalent (nucleophilic) catalysis — serine, cysteine, lysine, histidine, aspartate
• Metal ion & electrostatic catalysis — lysine, arginine, Mg²⁺, Zn²⁺ (stabilize charges)
• Transition-state stabilization — enzyme binds the transition state more tightly than the ground state, directly lowering ΔG‡

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PART B — FACTORS AFFECTING ENZYME ACTIVITY

1. 🌡️ Temperature

• Every 10°C rise in temperature approximately doubles enzyme activity
• Higher temperatures → faster reaction rates → better sensitivity at low enzyme concentrations
• Lower temperatures → extend the linear range of an assay (fewer dilutions needed)

Clinical relevance: Assay temperature must be maintained within ±0.1°C for accurate results. Storage of serum samples at −80°C preserves enzyme activity better than −20°C.

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2. 🧪 pH

• Each enzyme has a pH optimum for maximal activity; activity drops on either side of this optimum due to changes in ionization of active site residues and substrate
• Some enzymes have a broad pH optimum (less sensitive to small changes)
• Others have a narrow, sharp optimum

PharmD note: Drugs formulated for intestinal absorption must survive gastric pH (~2) before reaching enzymes active at intestinal pH (~7). This is why enteric coatings matter.

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3. 🔬 Substrate Concentration — Michaelis-Menten Kinetics

• At low [S]: reaction is first-order (rate ∝ [S])
• At high [S] (saturating): reaction is zero-order (rate = Vmax, independent of [S])
• This is called enzyme saturation — all active sites are occupied, and more substrate must wait

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4. 🚫 Enzyme Inhibitors

A. Irreversible Inhibition

• Inhibitor forms a permanent covalent bond with the enzyme → complete, non-recoverable loss of activity
• Example: organophosphates irreversibly inhibit acetylcholinesterase (used in nerve agents, insecticides)

B. Reversible Inhibition — 3 Types:

• Methotrexate (cancer drug) → inhibits dihydrofolate reductase
• ACE inhibitors (e.g., lisinopril, enalapril) → inhibit angiotensin-converting enzyme → lower blood pressure
• Malonate → inhibits succinate dehydrogenase (Krebs cycle)
• Fluoride → competitively inhibits enolase (glycolysis) — used in fluoride tubes to prevent glycolysis in blood samples

Competitive inhibition can be overcome by increasing substrate concentration. Irreversible inhibition cannot.

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5. ⚗️ Cofactors and Coenzymes

• Many enzymes require non-protein helpers to be active:

• An enzyme without its cofactor = apoenzyme (inactive)
• Apoenzyme + cofactor = holoenzyme (active)

PharmD relevance: Vitamin deficiencies → cofactor deficiency → enzyme malfunction → disease (e.g., Vit B1/thiamine deficiency → pyruvate dehydrogenase failure → Wernicke's encephalopathy)

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6. 🧬 Enzyme Concentration

• At constant [S], the reaction velocity is directly proportional to enzyme concentration
• This is the principle behind clinical enzyme assays — measuring how fast a reaction proceeds tells you how much enzyme is present in a patient's serum
• Elevated serum enzymes indicate tissue damage (e.g., elevated ALT/AST in liver disease; elevated CK in myocardial infarction)

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7. 🔗 Allosteric Regulation

• Some enzymes have regulatory (allosteric) sites separate from the active site
• Binding of activators or inhibitors at these sites causes conformational changes that alter Km or Vmax
• Allosteric enzymes often follow sigmoidal (not hyperbolic) kinetics
• These enzymes typically catalyze rate-limiting steps in metabolic pathways (e.g., phosphofructokinase in glycolysis)

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8. 🧫 Product Concentration (Product Inhibition)

• Accumulation of reaction products can inhibit the enzyme (often competitive, since product is structurally similar to substrate)
• This is called end-product inhibition — a physiological feedback mechanism
• High product concentration can also reverse the reaction, regenerating substrate

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Summary Table

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