CHEM 210
LO1 → Enzymology CHEM 210 → Clinical Chemistry 2
Enzymes
Enzymes are proteins that catalyze biochemical reactions.
Catalysts are substances that initiate and/or speed up chemical reactions without being consumed or altered in the reaction.
Enzymes are essential for various biochemical reactions, including:
DNA synthesis
Nerve conduction
Cell growth
Energy generation
Distribution of Enzymes
Enzymes are found within all body tissues.
They appear in serum following cellular injury or cell degradation.
Most biochemical reactions require energy input to ensure that molecules collide with sufficient energy, facilitating the reaction process.
Chemical Kinetics and Collision Theory
Collision theory states that a reaction occurs when molecules collide, allowing for bond formation.
Most biochemical reactions require additional energy to increase the speed of collisions, ultimately enhancing the reaction rate.
Activation Energy
Activation energy is the energy required to raise all molecules in 1 mole of a compound at a specific temperature to the transition state at the peak of the energy barrier.
If molecules possess sufficient energy, they will form products.
Enzymes lower the amount of free energy required to activate a reaction represented by the equation:
Enzyme reactions can occur in both directions, with the reaction rate in either direction proportional to the concentration of the reactants.
Enzyme Structure
Enzymes have structural levels:
Primary Structure: A sequence of amino acids.
Secondary Structure: Amino acids coil due to peptide bonds linking long rows.
Tertiary Structure: Coiled structures twist back on themselves through bonds between R groups.
Quaternary Structure: Involves the arrangement of multiple protein subunits, indirectly helping to identify tissue origin of the enzyme.
Isoenzymes
Isoenzymes are enzymes that differ in their relative proportions of different subunits but not in their catalytic function.
Act on the same substrate to produce identical products.
Example: AST from heart vs AST from liver can exhibit differences in ways such as affinity for substrates and inhibitors, immunological properties, and physical properties.
Active Sites and Specificity
Active site: The substrate for each enzyme must match the active site uniquely, with specific amino acid residues determining substrate specificity.
Absolute specificity: Some enzymes exhibit strict substrate specificity.
Group specificity: Others show broader specificity.
Enzyme Kinetics
When the substrate binds to the active site, it can undergo a structural change (shift) that leads to product formation, described by the formula:
An adduct is the enzyme-substrate complex formed during the process.
Allosteric site: A regulatory site that may influence enzyme activity and reaction rate.
Substrate Interaction
Substrates undergo several potential changes at the active site:
Change in the energy level of substrates
Addition of molecular groups
Removal of molecular groups
Physical alteration to produce products.
Enzyme Nomenclature
IUB (International Union of Biochemistry) established a systematic nomenclature for enzymes, typically naming them based on the substrate they act upon followed by the suffix “-ase.”
Example: Lipase acts on lipid substrates.
Classes of Enzymes
There are six main classes based on the chemical reactions that occur:
Oxidoreductases: Catalyze oxidation-reduction reactions.
Transferases: Transfer groups of atoms from one molecule to another.
Hydrolases: Addition or removal of water.
Lyases: Catalyze the breaking of bonds in molecules.
Ligases: Join two compounds at the cost of ATP or other energy sources.
Isomerases: Catalyze the conversion of one isomer to another (e.g., converting from high energy to low).
Factors Affecting Enzyme Activity
Factors influencing enzyme efficacy include:
Substrate concentration
Enzyme concentration
pH level
Temperature
Time
Presence of inhibitors (compounds that affect enzyme activity).
Presence of activators (compounds that enhance enzyme activity).
pH: Different enzymes function optimally at various pH levels, e.g. salivary amylase vs pancreatic amylase.
Extreme pH values may lead to protein denaturation.
Temperature: Increases in temperature generally boost reaction rates, with an increase of 10°C doubling the reaction rate until reaching denaturation.
Most enzymes have an optimal temperature below 40°C.
Time: Accurate timing is crucial as the rate depends on the quantity of substrate converted to product per minute.
Inhibitors
Inhibitors: Substances that interfere with enzyme reactions:
Competitive Inhibitors: Bind to active sites, reversible, effect decreases with increased substrate concentration.
Clinical example: Ethanol (ETOH) in ethylene glycol (EG) poisoning.
Non-competitive Inhibitors: Bind to an alternative site, not affected by substrate concentration.
Allosteric Inhibitors: Bind leading to structural changes, irreversible.
Example: Penicillin inhibits transpeptidase, essential for bacterial cell wall synthesis.
Uncompetitive Inhibitors: Bind to the enzyme-substrate complex.
Drugs as Enzyme Inhibitors
Examples:
ACE inhibitors (e.g., captopril): Inhibit angiotensin-converting enzyme (ACE), affecting vasoconstriction and hypertension.
Cofactors
Cofactors are non-protein molecules necessary for enzyme function, categorized into:
Activators: Inorganic ions (e.g., Mg, Ca, K, Zn, Fe).
Coenzymes: Organic molecules (e.g., NAD/NADH).
Michaelis-Menton Kinetics
Vmax: The maximum reaction rate when the enzyme is saturated with substrate.
Km: Michaelis constant, the substrate concentration at half of Vmax, indicating the enzyme's affinity for the substrate.
A small Km reflects a high affinity for the substrate.
LO2 → Enzymes in Serum
Enzyme assays typically involve a substrate present in excess. The amount of product formed is a function of enzyme concentration and catalytic activity.
Serum levels of enzymes can be diagnostic for various diseases. Organ damage leads to enzyme release into circulation, as seen in conditions like hepatitis or myocardial infarction. Enzyme levels can also rise due to increased cellular demands or drug therapies.
Enzyme analyses may include:
Measurement of product concentration.
Measurement of substrate concentration.
Measurement of coenzyme concentration.
Zero Order Kinetics: Enzyme concentration is the limiting factor; sufficient substrate and coenzymes lead to constant reaction rates, allowing continuous measurement of product formation.
Recovery of enzymatic activity through analysis is fundamental. For example, serum levels of certain enzymes can indicate liver, cardiac, or musculature dysfunction, aiding in diagnosing conditions like malignancies, pancreatitis, or hepatic disorders.
Quantifying Enzymes in Serum
Quantifying serum enzymes involves:
Detecting enzyme release from tissues in response to damage or necrosis.
Assessing via enzyme activity or catalytic reaction instead of direct concentration measurement.
Measuring rates can calculate enzyme levels based on product formation or substrate depletion.
Common Enzymes Measured
Common enzymes analyzed include:
ACP (Acid phosphatase)
ALT (Alanine aminotransferase)
ALP (Alkaline phosphatase)
Amylase
AST (Aspartate aminotransferase)
CK (Creatine kinase)
GGT (Gamma-glutamyltransferase)
LD (Lactate dehydrogenase)
Lipase
Specific Enzyme Tests
Each enzyme has specific diagnostic implications based on tissue source and condition:
ACP: Elevated in prostate cancer.
ALT: High levels indicate liver disease, stable when compared with AST.
AST: Elevated in myocardial infarction, liver, and muscle disorders.
ALP: Used in diagnosing osteomalacia, liver diseases; rises in pregnancy.
GGT: Sensitive to liver pathology, elevated in alcoholic liver diseases.
CK: Indicator for myocardial infarction and skeletal muscle disorders.
LD: Elevates in several pathological conditions including tissue damage.
Amylase and Lipase: Indicators of pancreatic function and conditions affecting the pancreas.
LO3 → Immunoassay Kits
Occult Blood Testing
Aims to detect hidden blood in stool, indicating possible malignancies or disorders.
Fecal Immunochemical Testing (FIT)
Utilizes antibodies for human hemoglobin detection.
Specific, sensitive, requiring no dietary changes for accuracy.
Infectious Mononucleosis Testing
Look for heterophile antibodies indicative of Epstein-Barr virus infection.
Differential diagnoses include evaluating leukocytosis and lymphocyte morphology.
Human Chorionic Gonadotropin (βHCG)
Hormone produced by the placenta, vital for pregnancy confirmation.
Tested for its specific β subunit for pregnancy verification.
Testing Methods & Considerations
Qualitative vs. quantitative methods for pregnancy detection and other analytes.
Limitations include false positives due to various tumor markers or physiological changes like ectopic pregnancies.
Innovacon Kit Methodology
Competitive immunoassay principles dictate binding of drug-label conjugates with specific antibodies, yielding readable results based on drug concentration.
Separation Techniques
Heterogeneous vs. Homogeneous Assays
Techniques involving free-labeled and bound-labeled reactants differ.
Sources of error include non-specific binding and antibody excess.
Immunoassay Specificity
Requires understanding antibody-antigen relationships, optimizing results, and avoiding interference from other substances.
Therapeutic Drug Monitoring (TDM)
Involves evaluating drug concentrations and mitigating toxic side effects, requiring strict adherence to timing and proper handling of specimens.
Factors affecting drug monitoring include:
Patient physiology
Drug interactions
Timing of sample collection (trough vs. peak levels).
Pharmacokinetics
Studies absorption, distribution, metabolism, and excretion affecting serum concentrations.
Reflects on drug behavior within the body, necessitating awareness of genetic factors that influence individual responses to therapy.