1. Enzymes
Part 1
M. Zaharna Clin. Chem. 2009

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
and without changing the equilibrium of the reaction.
A product is produced at the end of the reaction
M. Zaharna Clin. Chem. 2009

3. Introduction
The catalyzed reactions are frequently specific and essential to physiologic functions, such as:
the hydration of carbon dioxide,
nerve conduction,
muscle contraction,
nutrient degradation,
and energy use.
Found in all body tissue, enzymes frequently appear in the serum following cellular injury or, sometimes, in smaller amounts, from degraded cells.
M. Zaharna Clin. Chem. 2009

4. M. Zaharna Clin. Chem. 2009

5. 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 charged amino acid residues
Allosteric site
Another site on enzyme where co-factors or regulatory molecules interact
M. Zaharna Clin. Chem. 2009

6. Isoenzymes
Isoenzymes – are enzymes that differ in amino acid sequence but catalyze the same chemical reaction.
They have similar catalytic activity, but are different biochemically or immunologically.
Different forms may be differentiated from each other based on certain physical properties
electrophoretic mobility,
differences in absorption properties
or by their reaction with a specific antibody
These enzymes usually display different kinetic parameters (i.e. different KM values), or different regulatory properties. The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage
M. Zaharna Clin. Chem. 2009

7. Cofactors Non-protein molecules required for enzyme activation
Inorganic Activators
Chloride or magnesium ions, etc.
Organic Coenzymes
e.g. Nicotinamide adenine dinucleotide (NAD)
M. Zaharna Clin. Chem. 2009

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
Catalyze the joining of two substrate molecules
M. Zaharna Clin. Chem. 2009

9. M. Zaharna Clin. Chem. 2009

10. Enzyme classification
Plasma vs. non-plasma specific enzymes
Plasma specific enzymes have 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: cholinesterase, plasmin, thrombin
cholinesterase is an enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation.
M. Zaharna Clin. Chem. 2009

11. Enzyme classification
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.
An increased plasma concentration of these enzymes is associated with cell disruption or death
M. Zaharna Clin. Chem. 2009

12. 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
M. Zaharna Clin. Chem. 2009

13. Factors That Influence Enzymatic Reactions
Substrate Concentration
Enzyme Concentration pH
Temperature
Cofactors
Inhibitors
M. Zaharna Clin. Chem. 2009

14. Measuring enzyme activity
Enzymes are usually present in very small quantities in biologic fluids and often difficult to isolate from similar compounds
Therefore, Enzymes are not directly measured
Enzymes are commonly measured in terms of their catalytic activity
We don’t measure the molecule …
But we measure how much “work” it performs (catalytic activity)
That means 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
M. Zaharna Clin. Chem. 2009

15. Measuring enzyme 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.
M. Zaharna Clin. Chem. 2009

16. Measuring enzyme activity
NADH ( a common co-enzyme ) ( the reduced form ) absorbs light at 340 NM
NAD does not absorb light at 340 nm
Increased ( or decreased ) NADH concentration in a solution will cause the Absorbance ( A ) to change.
M. Zaharna Clin. Chem. 2009

17. Measurement Conditions
Excess amounts of substrate and any cofactors or coenzymes
to handle possible abnormally high patient enzyme levels
Proper temperature and pH
Inhibitors must be lacking
The temperature should be constant within ±0.1°C throughout the assay at a temperature at which the enzyme is active
M. Zaharna Clin. Chem. 2009

18. Methods for Enzyme measurement
Fixed time methods
the reactants are combined,
the reaction proceeds for a designated time,
the reaction is stopped (usually by inactivating the enzyme with a weak acid),
a measurement is made of the amount of reaction that has occurred.
The reaction is assumed to be linear over the reaction time; the larger the reaction, the more enzyme is present.
Possible problems with extremely high enzyme levels
M. Zaharna Clin. Chem. 2009

19. Methods for Enzyme measurement
Continuous-monitoring methods
multiple measurements, usually of absorbance change, are made during the reaction,
either at specific time intervals (usually every 30 or 60 seconds)
or continuously by a continuous-recording spectrophotometer.
These assays are advantageous over fixed-time methods because the linearity of the reaction may be more adequately verified.
M. Zaharna Clin. Chem. 2009

20. Methods for Enzyme measurement
Continuous-monitoring methods
If absorbance is measured at intervals, several data points are necessary to increase the accuracy of linearity assessment.
Continuous measurements are preferred because any deviation from linearity is readily observable.
M. Zaharna Clin. Chem. 2009

21. 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
International System of Units
M. Zaharna Clin. Chem. 2009

22. Measurement of Enzyme Mass
Immunoassay methodologies that quantify enzyme concentration by mass are also available and are routinely used for quantification of some enzymes.
Immunoassays may overestimate active enzyme as a result of:
possible cross-reactivity with inactive enzymes,
inactive isoenzymes,
or partially digested enzyme.
M. Zaharna Clin. Chem. 2009

23. Measurement of Enzyme Mass
The relationship between enzyme activity and enzyme quantity is generally linear but should be determined for each enzyme.
Enzymes may also be determined and quantified by electrophoresis techniques which provide resolution of isoenzymes.
M. Zaharna Clin. Chem. 2009

24. M. Zaharna Clin. Chem. 2009

25. Creatine Kinase (CK)
Action – This enzyme is associated with the regeneration and storage of high energy phosphate (ATP).
It catalyzes the following reversible reaction in the body.
M. Zaharna Clin. Chem. 2009

26. Creatine Kinase (CK) High concentrations of CK in:
skeletal muscle,
cardiac muscle
and brain tissue
Increased plasma CK activity is associated with damage to these tissues
 CK is especially useful to diagnose:
Acute Myocardial Infarction (AMI)
Skeletal muscle diseases ( Muscular Dystrophy )
Muscular dystrophy (MD) refers to a group of genetic, hereditary muscle diseases that weaken the muscles that move the human body.[1][2] Muscular dystrophies are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue
M. Zaharna Clin. Chem. 2009

27. Creatine Kinase (CK) CK has 3 isoenzymes
Each isoenzyme is composed of two different polypeptide chains (M & B)
CK - BB (CK1) Brain type
CK - MB (CK2) Cardiac type or hybrid type
CK – MM (CK3) Muscle type
Normal serum consists of approximately 94% to 100% CK-MM
Cardiac muscle CK is 80% CK-MM and 20% CK-MB
M. Zaharna Clin. Chem. 2009

28. Creatine Kinase (CK) BB migrates fastest to anode than MB & MM
The major isoenzyme in the sera of healthy people is the MM form.
Values for the MB isoenzyme range from undetectable to trace (<6% of total CK).
It also appears that CK-BB is present in small quantities in the sera of healthy people
M. Zaharna Clin. Chem. 2009

29. Diagnostic Significance
The value of CK isoenzyme separation can be found principally in detection of myocardial damage.
Cardiac tissue contains significant quantities of CK-MB, approximately 20% of all CK-MB.
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 hours post AMI
Returns to normal hours
M. Zaharna Clin. Chem. 2009

30. M. Zaharna Clin. Chem. 2009

31. CK Assay CK assays are often coupled assays.
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
M. Zaharna Clin. Chem. 2009

32. CK Assay Reference Range for Total CK: Male, 15-160 U/L (37°C)
Female, U/L (37°C)
CK-MB: <6% total CK
M. Zaharna Clin. Chem. 2009

33. CK isoenzymes
For CK isoenzymes, electrophoresis is the reference method.
Other methods include ion-exchange chromatography, and radioimmunoassay.
Rapid assay for CK-MB subforms, uses high voltage electrophoresis on an automated analyzer, the result will be available in 25 min.
M. Zaharna Clin. Chem. 2009

34. Lactate Dehydrogenase (LD)
Catalyzes interconversion of lactic and pyruvic acids
It is a hydrogen-transfer enzyme
NAD is used as coenzyme
High activities in heart, liver, muscle, kidney, and RBC
Lesser amounts: Lung, smooth muscle and brain
M. Zaharna Clin. Chem. 2009

35. 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 ( HHHH ) Cardiac , RBCs
LD ( HHHM ) Cardiac , RBCs
LD ( HHMM ) Lung, spleen, pancreas
LD ( HMMM ) Hepatic
LD ( MMMM ) Skeletal muscle
LD-1 is the fastest towards the anode
M. Zaharna Clin. Chem. 2009

36. Diagnostic Significance
LDH is elevated in a variety of disorders.
in cardiac,
hepatic,
skeletal muscle,
and renal diseases,
as well as in several hematologic and neoplastic disorders
The highest levels of LD-1 are seen in pernicious anemia and hemolytic disorders
LD-3 with pulmonary involvement
LD-5 predominates with liver & muscle damage
M. Zaharna Clin. Chem. 2009

37. Diagnostic Significance
In healthy individuals
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
M. Zaharna Clin. Chem. 2009

38. Diagnostic Significance
A sixth LDH isoenzyme has been identified
LDH-6 has been present in patients with arteriosclerotic cardiovascular failure
Its appearance signifies a grave prognosis and impending death
It is suggested, that LDH-6 may reflect liver injury secondary to severe circulatory insufficiency
M. Zaharna Clin. Chem. 2009

39. Assay for Enzyme activity
The reaction can proceed in either a forward or reverse direction
Pyruvate + NAD+ Lactate + NADH + H+
The optimal pH:
for the forward reaction is 8.3 – 8.9
For the reverse reaction 7.1 – 7.4
Reference Range : U/L (37°C) LD
M. Zaharna Clin. Chem. 2009

40. Aspartate Aminotransferase (AST, SGOT, GOT)
Transferase class of enzymes - transaminase
Transaminase involved in the transfer of an amino group between aspartate and -ketoacids.
Pyridoxal phosphate is coenzyme
Source is heart, liver, and skeletal muscle
Serum Glutamic Oxalocetic Transaminase – SGOT
The older terminology, serum glutamic-oxaloacetic transaminase (SGOT, or GOT)
M. Zaharna Clin. Chem. 2009

41. Aspartate Aminotransferase (AST, SGOT, GOT)
The transamination reaction is important in intermediary metabolism because of its function in the synthesis and degradation of amino acids.
The ketoacids formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle to provide a source of energy.
M. Zaharna Clin. Chem. 2009

42. Diagnostic Significance
The clinical use of AST is limited mainly to the evaluation of hepatocellular disorders and skeletal muscle involvement.
Post AMI
Rises 6 – 8 hours
Peaks at 24 hours
Returns to normal by day 5
AST levels are highest in acute hepatocellular disorders, viral hepatitis, cirrhosis.
Viral hepatitis may reach 100 x ULN
M. Zaharna Clin. Chem. 2009

43. Diagnostic Significance
There are two isoenzyme fractions located in the cell cytoplasm and mitochondria,
the cytoplasmic isoenzyme is predominant in serum
while the mitochondrial one may be increased following cell necrosis.
Isoenzyme analysis of AST is not routinely performed in the clinical laboratory.
M. Zaharna Clin. Chem. 2009

44. Assay for Enzyme activity
Measurement by Karmen method – use Malate dehydrogenase in second step
Detect change in absorbance at 340 nm
Aspartate + -Ketoglutarate Oxaloacetate + Glutamate
Oxaloacetate + NADH + H Malate + NAD MD
AST
Reference Range : 5 to 30 U/L (37°C)
M. Zaharna Clin. Chem. 2009