Enzyme Overview and Nomenclature

Enzyme Overview

• Enzymes are specific proteins that catalyze biochemical reactions.
• They lower the activation energy needed for reactions to proceed.
• Enzymes are not consumed or altered during the reaction.
• They are regenerated and act on a substrate.
• They can be found in all body tissues.
• Enzyme levels in the serum can increase after cellular injury.
• Enzymes are proteins composed of specific amino acid sequences.
• They can be denatured by heat and changes in pH.
• Isoenzymes and isoforms are different forms of the same enzyme.

Enzyme Nomenclature

• The Enzyme Commission (EC) developed a classification system for enzymes in 1961.
• Systematic names describe the substrate, reaction, and coenzyme.
• Recommended names are more practical.
• Abbreviations are used in laboratories.
• Code numbers are four-digit numbers separated by decimal points.
• The first digit places the enzyme into one of six classes.

Enzyme Classes

• Oxidoreductases catalyze oxidation-reduction reactions between two substrates.
• Transferases transfer a group other than hydrogen from one substrate to another.
• Hydrolases catalyze hydrolysis of various bonds.
• Lyases remove groups from substrates without hydrolysis.
• Isomerases interconvert geometric, optical, or positional isomers.
• Ligases catalyze the joining of two substrate molecules.

Clinically Significant Enzymes

• Lactate dehydrogenase (LDH)
• Aspartate aminotransferase (AST)
• Alanine aminotransferase (ALT)
• Creatine kinase (CK)
• Gamma glutamyltransferase (GGT)
• Alkaline phosphatase (ALP)
• Acid phosphatase (ACP)
• Amylase (AMS/AMY)

Enzyme Activity Measurement

• Measuring enzyme activity is common and relates to enzyme concentration.
• Enzyme concentrations in blood are normally low.
• Increased levels indicate disease.
• Immunoassays can detect some enzymes directly.
• Enzyme concentration can be measured by mass (e.g., Creatine Kinase, CK).
• Electrophoresis is used to measure isoenzymes or isoforms.
• Different methods for measuring enzyme activity exist.

Enzyme Kinetics

• Enzyme activity can be measured by changes in product concentration, substrate concentration or coenzyme concentration.
• The initial phase of the reaction may exhibit zero-order kinetics.
• Zero-order kinetics depend only on enzyme concentration, not on substrate concentration.
• If other variables affect the rate (e.g., substrate depletion), then First-order kinetics exist.
• Measurements are done to specific time points and are either 2-point (or fixed time) assays or kinetic assays.
• Enzyme concentration can be very high in a sample. If this occurs the reaction won't be linear, the sample can be diluted and reanalyzed.

Michaelis-Menton Curve

• Km is the Michaelis constant (substrate concentration at half maximum velocity).
• Max velocity is the maximum rate of reaction.
• Vmax is the maximum reaction velocity.
• Manufacturers will ensure that substrate concentration is greater than Km so zero order kinetics apply to the assay.

Factors Influencing Enzyme Activity

• Substrate concentration ensures that the substrate does not run out.
• Reaction should be buffered at an optimal pH.
• Changes in pH can denature enzymes.
• Physiologic enzymatic reactions occur between pH 7.0 and 8.0.
• Temperature affects enzyme activity.
• Increased temperature increases activity until too high leading to denaturation.
• Rate of reaction doubles with every 10°C increase (Q10).
• Enzyme concentration directly relates to reaction speed (higher concentration = faster reaction).

Cofactors

• Cofactors are nonprotein molecules that bind to enzymes.
• They are required for some enzymes to function.
• Inorganic cofactors include metals (e.g., Ca2+, Fe2+, Mg2+) and nonmetals (e.g., Br, Cl-).
• Organic cofactors include coenzymes (phosphates and vitamins).

Inhibitors & Activators

• Activators increase the rate of reaction.
• Inhibitors decrease the rate of reaction.

Enzyme Terms

• Coenzyme: an organic cofactor.
• Prosthetic group is a tightly bound coenzyme.
• Holoenzyme is an active enzyme with all required components.
• Apoenzyme is the protein portion of an enzyme without any cofactors.
• Proenzyme (zymogen): an inactive enzyme form.
• The inactive form of an enzyme that can be converted into active enzyme through specific chemical processes, mainly in digestive enzymes.

Enzyme Reporting (Activity)

• The international unit (IU) is used to report enzyme activity.
• 1 IU is the amount of enzyme needed to catalyze the reaction of 1 µmol of substrate per minute per liter.
• The results are reported at IU/L or mIU/mL..

Enzymes as Reagents

• Enzymes are used to measure non-enzymatic substances.
• Glucose oxidase is used for glucose measurement, and uricase measures uric acid.

Clinical Significance & Sources of Error

• Enzymes produced in cells can be found in plasma or fluids.
• Increased enzymes can indicate disease, (ex. increased production, or leakage) of the tissue or organ.
• Hemolyzed samples should be avoided since enzymes are higher in red blood cells.
• Measurements should be made ASAP to prevent an increase due to standing.
• Increased triglycerides are a source of error, which can falsely diminish enzyme measurement values.
• Some enzymes, such as acid phosphatase, are unstable at room temperature and must be measured quickly.

Isoenzymes

• Isoenzymes are different forms of the same enzyme.
• They have the same active site, catalyzing the same reaction.
• They differ in their amino acid structure.
• Isoenzymes can come from different tissues, such as Liver, bone or organs.
• Electrophoresis and immunochemical methods can be used for identification.

Methods to Identify Isoenzymes

• Zone electrophoresis (AKA protein electrophoresis) is used, Enzymes have different protein structures.
• Selective inactivation of enzymes allows specific isoenzymes to be examined.
• Increased temperature or chemical inactivation will denote specific isoenzyme from other inactive isoforms.
• Immunochemical methods use antisera to identify specific isoenzymes.