Enzymes

1. Enzymes

2. Introduction • Enzymes are usually proteins that act as catalysts, compounds that increase the rate of chemical reactions. • They bind specifically to a substrate, forming a complex. • This complex lowers the activation energy in the reaction: • without the enzyme becoming consumed or • without changing the equilibrium of the reaction. • A product is produced at the end of the reaction • Mohammed Laqqan

3. General Properties of Enzymes • Like all proteins 1°, 2°, 3°, and 4° structures • Active site → cavity where substrate interacts • Often water-free site • Reacts with charge moieties • Allosteric site • Another site on enzyme where co-factors or regulatory molecules interact • Mohammed Laqqan

4. Isoenzyme • Isoenzymes: are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. • Now called “isoform” of an enzyme • They have similar catalytic activity, but are different biochemically or immunologically and can be demonstrated by electrophoretic mobility, differences in absorption properties or by their reaction with a specific antibody • Mohammed Laqqan

5. Cofactors • Non-protein molecules required for enzyme activation • Inorganic Activators • Chloride or magnesium ions, etc. • Organic coenzymes • e.g. Nicotinamide adenine dinucleotide (NAD) • Mohammed Laqqan

6. Enzyme classification Plasma vs. non-plasma specific enzymes • Plasma specific enzymeshave a very definite/specific function in the plasma 1) Plasma is normal site of action 2) Concentration in plasma is greater than in most tissues 3) Often are liver synthesized 4) Examples: plasmin, thrombin • Mohammed Laqqan

7. Non-plasma specific enzymes have no known physiological function in the plasma 1) Some are secreted into the plasma 2) A number of enzymes associated with cell metabolism normally found in the plasma only in low concentrations. • Source of non-plasma enzymes • From cells during the normal process of breakdown and replacement. • higher concentration following injury or death of tissue cells. • altered membrane permeability that may occur with inflammation An increased plasma concentration of these enzymes is associated with cell disruption or death • Mohammed Laqqan

8. Classes of Enzymes • International Union of Biochemistry (IUB) 1 = Oxidoreductases (Examples: LDH, G6PD) Involved in oxidation - reduction reactions 2 = Transferases (Examples: AST, ALT) Transfer functional groups 3 = Hydrolases (Examples: acid phosphatase, lipase) Transfer groups to -OH 4 = Lyases (Examples: aldolase, decarboxylases) Add across a double bond 5 = Isomerases (Example: glucose phosphate isomerase) Involved in molecular rearrangements 6 = Ligases Complicated reactions with ATP cleavage • Mohammed Laqqan

9. Factors Affecting Enzyme Levels in Blood • Entry of enzymes into the blood • Leakage from cells • Altered production of enzymes • e.g. increased osteoblastic activity results in increase in enzymes in bone disease • Clearance of enzymes • Half life vary from few hours to several days • Mohammed Laqqan

10. Enzyme measurement • Enzymes are not directly measured • Enzymes are commonly measured in terms of their catalytic activity • We don’t measure the molecule … • We measure how much “work” it performs (catalytic activity) • The rate at which it catalyzes the conversion of substrate to product • The enzymatic activity is a reflection of its concentration • Activity is proportional to concentration • Mohammed Laqqan

11. Photometric measurement of activity • Enzyme activity can be tested by measuring • Increase of product • Decrease of substrate • Decrease of co-enzyme • Increase of altered co-enzyme • If substrate and co-enzyme are in excess concentration, the reaction rate is controlled by the enzyme activity. • Mohammed Laqqan

12. Measuring enzyme activity • NADH( a common co-enzyme ) absorbs light at 340 NM • NAD ( the reduced form ) does not absorb light at 340 nm • Increased ( or decreased ) NADH concentration in a solution will cause the Absorbance (Abs) to change. • Mohammed Laqqan

13. Measurement of Enzymatic Activity • One of two general methods may be used to measure the extent of an enzymatic reaction • Fixed time • Measure at specified time (e.g. 0 and 60 seconds) • The reactants are combined the reaction proceed for a designated time • The reaction is stopped usually by inactivating the enzyme with a weak acid • And a measurement is made of the amount of reaction that has occurred • Mohammed Laqqan

14. Measurement of Enzymatic Activity • Continuous monitoring or kinetic assay • Are recorded by the spectrophotometer • Measurements of absorbance change are made during the reaction either at: • Measure at specific time intervals (usually every 0, 30, 60, 90, 120 seconds) • Mohammed Laqqan

15. Measurement Units • Reported as “activity” not concentration • IU = amount of enzyme that will convert 1 μmol of substrate per minute in specified conditions • Usually reported in IU per liter (IU / L) • SI unit = Katal = mol/sec moles of substrate converted per second • enzyme reported as katals per liter (kat / L) • 1 IU = 17nkat • Mohammed Laqqan

17. Creatine Kinase (CK) • Action of this enzyme is associated with the regeneration and storage of high energy phosphate (ATP). • The enzyme catalyzes the conversion of Creatine to Creatine Phosphate • The enzyme also catalyzes the reversible reaction • Found in skeletal muscle, cardiac muscle, and brain • CK is especially useful to diagnose • AMIs • Skeletal muscle diseases ( Muscular Dystrophy ) • Mohammed Laqqan

18. CK has 3 isoenzymes • Each isoenzyme is composed of two different polypeptide chains (M & B) • CK - BB (CK1) Brain • CK - MB (CK2) Cardiac • CK - MM (CK3) Muscle • Skeletal muscle CK is 99% CK-MM • Cardiac muscle CK is 80% CK-MM and 20% CK-MB • BB migrates fastest to anode then MB & MM • MM is highest in serum in healthy patients • MB trace to <6% total, BB 0-trace • Mohammed Laqqan

19. Because of CK – MB’s association with cardiac tissue, • increased CK – MB ( > 6% of the total CK activity ) is a strong indication of AMI • Post AMI CK-MB • CK-MB increases 4 – 8 hours post AMI • Peaks at 12 - 24 hours post AMI • Returns to normal 48 - 72 hours later

20. Specimen: Serum, heparin plasma or EDTA plasma. • CK assays are often coupled assays CK • In the example below, the rate at which NADPH is produced is a function of CK activity in the first reaction. • Hexokinase and G6PD are auxiliary enzymes • Reverse reaction most commonly performed in clinical laboratory methods

21. Interference • RBCs lack CK, but hemolyzed RBCs release Adenylate Kinase (AK) into the plasma, • AK reacts with ADP to produce ATP which is then available to participate in the reaction causing falsely increased CK activity • The interference can occur with hemolysis greater than 200 mg/dl of hemoglobin • CK should be stored in the dark place because CK is inactivated by daylight • Mohammed Laqqan

22. Lactate Dehydrogenase (LDH) • Catalyzes interconversion of lactic and pyruvic acids • NAD as coenzyme • High activities in heart, liver, muscle, kidney, and RBC • Lesser amounts: Lung, smooth muscle and brain • Elevated with diseases of the above (Liver disease, AMI & Hemolytic diseases) • Mohammed Laqqan

23. LDH Isoenzymes • Because increased total LDH is relatively non-specific, LDH isoenzymes can be useful • 5 isoenzymes composed of a cardiac (H) and muscle ( M ) component • LD - 1 ( HHHH ) Cardiac , RBCs • LD - 2 ( HHHM ) Cardiac , RBCs • LD - 3 ( HHMM ) Lung, spleen, pancreas • LD - 4 ( HMMM ) Hepatic and skeletal • LD - 5 ( MMMM ) Hepatic and skeletal • Mohammed Laqqan

24. In healthy patients • LD-2 is in highest quantity then LD-1, LD-3, LD-4 and LD-5 • Heart problems 2-10 x (Upper Limit of Normal) ULN in acute MI • If problem is not MI, both LD1 and LD2 rise, with LD2 being greater than LD1 • If problem is MI, LD1 is greater than LD2. This is known as a flipped pattern • The highest levels of LD are seen in pernicious anemia and hemolytic disorders • LD-3 with pulmonary involvement • LD-5 predominates with liver & muscle damage • Mohammed Laqqan

25. Assay for Enzyme activity • The reaction can proceed in either a forward or reverse direction • Lactate + NAD+ Pyruvate + NADH + H+ • The optimal pH: • for the forward reaction is 8.3 – 8.9 • For the reverse reaction 7.1 – 7.4 LD LD • Mohammed Laqqan

26. Specimen: Serum, heparin plasma or EDTA plasma • Measurements & Sources of error • RBCs have 100+ times the amount of LD • Hemolysis ruins sample for testing • LD unstable during storage – test within 48 hours • LD-5 most labile store at 25oC not 4oC • In AMI, LDH levels begin to rise within 12h to 24h. • Reach peak within 48h to 72h. • Remain elevated for 10 days. • Mohammed Laqqan

27. Clinical Significance • The measurement of the serum concentration of LD has proven to be useful in the diagnosis ofmyocardial infarction. • The LD enzyme activity in serum does not rise as much as CK or AST aftermyocardial infarction, but it does remain elevated for a much longer period of time. • This is quite important when the patient does not see a physician for 3 or 4 days following an infarct. • In hepatocellular disease, the serum activity of LD rises, but the measurement of this enzyme is much less useful than that of AST or ALT because the test is less sensitive. • Mohammed Laqqan