Enzymes Part 1: Liver, Pancreas & Cardiac

Enzymes - Part 1 (Liver, Pancreas, & Cardiac)

• Enzymes are specific biological proteins that catalyze biochemical reactions.
• They lower the activation energy needed for a reaction to proceed.
• Enzymes are not consumed or changed in composition during a reaction, and are regenerated.
• They act upon a substrate.
• Enzymes can be found in all body tissues and frequently appear in serum following cellular injury.
• Enzymes are proteins made of specific amino acid sequences.
• They can be denatured by heat, changes in pH, etc.
• Enzymes may exist in different forms – isoenzymes and isoforms.

Enzyme Nomenclature

• The Enzyme Commission (EC) developed a classification system in 1961.
• This system includes a systematic name, a recommended name, an abbreviation, and a code number.
• The systematic name defines the substrates acted on, the reaction catalyzed, and the name of any coenzymes.
• Recommended names are more usable/practical.
• Abbreviations are widely used in labs.
• The code number is a four-digit number separated by decimal points, with the first digit placing enzymes into one of six classes.

Classes of Enzymes

• The International Union of Biochemistry (IUB) system assigns a name and code to each enzyme.
• Oxidoreductases catalyze oxidation-reduction reactions between two substrates.
• Transferases catalyze the transfer of a group (excluding hydrogen) from one substrate to another.
• Hydrolases catalyze hydrolysis of various bonds (adding water). Substrates are broken down.
• Lyases catalyze the removal of groups from substrates without hydrolysis. The product will have double bonds.
• Isomerases catalyze the interconversion of geometric, optical, or positional isomers. (e.g., converting D to L forms)
• Ligases catalyze the joining of two substrate molecules, coupled with the breaking of a pyrophosphate bond in ATP.

Measurement of Enzyme Activity

• Enzyme concentrations in blood are normally very low. Increased levels indicate a disease process.
• Immunoassays can be used to detect enzymes directly.
• Enzyme concentration can be measured by mass (e.g., Creatine Kinase, CK).
• Electrophoretic techniques can be used to measure isoenzymes or isoforms.
• Enzyme activity can be measured by changes in product concentration, concentration of altered coenzyme, or decrease in substrate concentration.

Enzyme Kinetics

• Enzyme kinetics is studied in three phases.
  • Lag Phase; sample and reagents are mixed, and equilibrium is established, but it is non-linear
  • Linear Phase; product formation and substrate consumption are consistent and the reaction follows a zero-order rate. This is the phase for measuring
  • Substrate Depletion; product formation has slowed (not linear), this is not good for measuring when enzyme concentration is very high.
• If the enzyme concentration is significantly high, the reaction rate may not remain linear - substrate will be used up. The sample can be diluted and reanalyzed.

Methods for Reading Enzyme Reactions

• 2-Point Assay (Fixed time) involves readings at fixed times, usually an initial and a final reading. This is less accurate; it may not be during the linear phase.
• Kinetic Assay (Continuous Monitoring) takes multiple absorbance readings as the reaction proceeds (every 30–60 seconds or continuously). This is a more accurate method. Deviations from linearity can be detected.

Zero-Order Kinetics

• During the linear phase of enzyme kinetics.
• The reaction rate depends only on enzyme concentration.
• An increase in substrate does not change the reaction rate.
• Lab enzyme procedures mostly follow zero-order kinetics.
• First-order kinetics may be seen if other factors affect the rate (for example, substrate depletion).

Michaelis-Menton Curve

• Km represents the substrate concentration (Michaelis constant) at which the reaction velocity is half of its maximum.
• Vmax is the maximum velocity of the enzyme-catalyzed reaction. Manufacturers of assay kits ensure the substrate concentration is 10 to 100 times greater than the Km value to prevent substrate exhaustion and maintain zero-order kinetics.

Factors that Influence Enzyme Activity

• Substrate Concentration: ensure sufficient substrate is present to avoid running out.
• pH: Enzymes have an optimal pH for activity. Maintain appropriate buffers/solutions.
• Temperature: Increases in temperature often increase enzyme activity up to a point. However, beyond this point the enzyme will become denatured. Normal temperatures for tests and assay conditions will be at 37 degrees Celcius or 25/30 degrees Celcius.
• Enzyme Concentration: Higher enzyme concentration generally results in a faster reaction.

Cofactors

• Nonprotein molecules that bind to enzymes to facilitate their activity.
• Inorganic cofactors include metals (Ca2+, Fe2+, Mg2+) and nonmetals (Br-, Cl-).
• Coenzymes are organic cofactors that often contain vitamins or phosphates.

Inhibitors and Activators

• Activators increase reaction rate
• Inorganic cofactors are necessary for reaction mechanisms
• Nonmetallic and metallic ions act as cofactors.
• Inhibitors cause reactions to decrease in rate
• Inhibitors may be reversible or irreversible

Enzyme Terms

• Coenzyme: An organic cofactor (e.g., vitamins, NAD); not tightly bound.
• Prosthetic group: A tightly bound coenzyme/ cofactor
• Holoenzyme: active enzyme complex
• Apoenzyme: inactive enzyme portion without a cofactor.
• Proenzyme/Zymogen: an inactive enzyme form that is activated by other enzymes.

How Do We Report Enzyme Activity?

• Historically, various units were used for different methods.
• The current standard is the International Unit (IU).
• One IU is defined as the amount of enzyme that catalyzes the reaction of 1 μmol of substrate per minute per liter of serum.
• Units are written as IU/L or mIU/mL.

Enzymes as Reagents

• Enzymes can be used as reagents to measure non-enzymatic substances in serum.
• For example, Glucose oxidase and Uricase.

Enzyme Analysis vs. Enzymes As Reagents

• In enzyme analysis (measuring enzyme activity), substrate must be in excess (zero order), meaning rate depends on enzyme concentration.
• In measuring another substrate with an enzyme in excess (first-order), the reaction rate depends on substrate concentration.

Why Measure Enzymes?

• Enzymes are produced within cells and found in all body tissues.
• Plasma-specific enzymes function within the blood stream (e.g. coagulation)
• Cellular enzymes are released from damaged cells.
• Increased enzyme levels in the blood indicate disease or damage to the organ where the enzyme is produced. Increased enzyme levels may come from increased production from the organ or they could be due to damage of the organ/tissue leading to a leakage of enzymes into the blood stream

Isoenzymes

• Isoenzymes are different forms of the same enzyme with slightly different amino acid sequences and structures.
• They catalyze the same reaction but may have different tissue origins and heat stabilities.
• Isoenzymes can be differentiated via electrophoresis or selective inactivation techniques.

Methods to Identify Isoenzymes

• Zone Electrophoresis (protein electrophoresis) separates enzymes based on different protein structures. Visible bands are created when exposed to specific chemicals.
• Selective Inactivation; chemicals can denature or inhibit certain isoenzymes
• Immunochemical methods; specific antibodies (antisera) can identify specific isoenzymes.

Clinically Significant Enzymes (Chemistry)

• Alkaline Phosphatase (ALP)
• Acid Phosphatase (ACP)
• Amylase (AMS)
• Aspartate Transaminase (AST)
• Alanine Aminotransferase (ALT)
• Gamma Glutamyltransferase (GGT)
• Lactate Dehydrogenase (LDH)
• Creatine Kinase (CK)

Alkaline Phosphatase (ALP)

• Catalyzes the reaction of organic phosphate & water into an alcohol and phosphate.
• Nomenclature: Hydrolase class, Alkaline Phosphatase practical name, ALP abbreviation
• Optimum pH around 9–10
• Activators: Mg2+
• Inhibitors: oxalate, phosphate, borate, cyanide
• Four major isoenzymes (Bone, Liver, Intestine, and Placenta).

Alkaline Phosphatase (ALP) - Clinical Significance

• Used to assess liver and bone disorders
• Increased levels associated with: extrahepatic/intrahepatic obstruction,bone disease/cancer, Paget's Disease, certain pregnancy stages, and healing bone fractures.
• Decreased levels may indicate hypophosphatasia or bone calcification problems.
• Avoid hemolyzed samples and analyze ASAP.

Acid Phosphatase (ACP)

• Catalyzes the reaction of organic phosphate & water into an alcohol and phosphate.
• Nomenclature: Hydrolase class, Acid Phosphatase practical name, ACP abbreviation
• Optimum pH around 5
• Activators: Mg2+
• Inhibitors: oxalate, phosphate, borate, cyanide
• Highest concentration in prostate, several isoenzymes found in liver, bone, spleen, and urine.

Acid Phosphatase (ACP) - Clinical Significance

• Used to evaluate prostate carcinoma
• Increased levels in metastatic prostate cancer. Used in forensic investigations
• May appear in vaginal washings after rape where it is detected for up to 4 days
• May also increase due to bone disorders or platelet damage.

Acid Phosphatase (ACP) - Sources of Error

• ACP is unstable and must be tested immediately, otherwise activity will decrease.
• Separate serum from red blood cells and platelets to prevent a false increase
• If not measured immediately, freeze or use an acidifying buffer.

Prostate Specific Antigen (PSA)

• PSA is a serine protease secreted by prostatic epithelial cells.
• PSA is more sensitive and accurate than PAP for diagnosing prostate cancer.
• Monitoring for treatment success requires a baseline PSA.
• Increased PSA can indicate prostate cancer, inflammation, or other conditions/benign conditions

Amylase (AMS/AMY)

• Amylose and Amylopectin are linear and branched polyglucans
• Amylase hydrolyzes a-1,4-glycosidic linkages in the polymer
• Nomenclature: Hydrolase class, Amylase practical name, AMS/AMY abbreviation.
• Activators: Ca2+, Cl-
• Inhibitors: oxalate, citrate, fluoride.
• Amylase is frequently measured in patients with suspected pancreatic disease

Amylase - Clinical Significance

• Elevation is associated with acute pancreatitis,mumps, severe burns, and alcoholism
• Increased with salivary gland lesions, biliary tract disease

Amylase - Sources of Error

• Amylase is fairly stable. Little is lost after 1 week at room temperature or 2 months at 4°C
• However, high triglycerides can suppress amylase activity, making acute pancreatitis appear normal or falsely decreased.

Lipase

• Lipase breaks down lipids in the intestines.
• Lipase levels are commonly measured alongside amylase in cases of acute pancreatitis.

Isoenzymes of Amylase (AMS/AMY)

• Amylase isoenzymes (p type-pancreatic & s type-salivary) can be separated via electrophoresis, chromatography, or isoelectric focusing.

This quiz explores enzymatic functions and characteristics, focusing on the liver, pancreas, and cardiac enzymes. Learn about enzyme catalysis, nomenclature, and the importance of enzymes in various tissues. Test your knowledge on isoenzymes and enzyme denaturation.