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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
General Properties of Enzymes
• Like all proteins 1°, 2°, 3°,
and 4° structures
• Active site → cavity where
substrate interacts
• Allosteric site
– Another site on enzyme
where co-factors or
regulatory molecules
interact
Mohammed Laqqan
M. Zaharna Clin. Chem. Lab
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
M. Zaharna Clin. Chem. Lab
Enzyme Cofactors
1. Simple Enzymes: only protein chain(s)
2. Complex Enzymes: require additional molecules (co-factors)
for functioning
 Co-enzyme: non-covalently bound
 Prosthetic Group: covalently bound to protein
Prosthetic groups/co-enzymes usually VITAMINS!
Vitamin deficiency diseases: malfunction of enzymes
M. Zaharna Clin. Chem. Lab
Enzyme 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
M. Zaharna Clin. Chem. Lab
Enzyme classification
Plasma vs. non-plasma specific enzymes
a.
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: plasmin, thrombin
Mohammed Laqqan
M. Zaharna Clin. Chem. Lab
B.
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
M. Zaharna Clin. Chem. Lab
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 (Add water across a bond, hydrolyzing it)
4 = Lyases (Examples: aldolase, decarboxylases)
Add water, NH2, or CO2 across a double bond or remove these
elements to produce double bonds.
5 = Isomerases (Example: glucose phosphate isomerase)
Involved in molecular rearrangements
6 = Ligases Complicated reactions with ATP cleavage
Mohammed Laqqan
M. Zaharna Clin. Chem. Lab
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
M. Zaharna Clin. Chem. Lab
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
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
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
M. Zaharna Clin. Chem. Lab
Measurement of Enzymatic Activity
•
One of two general methods may be used to measure the
extent of an enzymatic reaction:
1.
Fixed time
–
Measure at specified time (e.g. 0 and 60 seconds).
2. Continuous monitoring or kinetic assay
–
Measure at specific time intervals (usually every 0, 30, 60,
90, 120 seconds)
Mohammed Laqqan
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
M. Zaharna Clin. Chem. Lab
M. Zaharna Clin. Chem. Lab
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
• Acute myocardial infarction (AMI)
• Skeletal muscle diseases ( Muscular Dystrophy )
Mohammed Laqqan
• 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
M. Zaharna Clin. Chem. Lab
• 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
M. Zaharna Clin. Chem. Lab
• 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
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
Lactate Dehydrogenase (LDH)
• Catalyzes interconversion of lactic acid 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
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
M. Zaharna Clin. Chem. Lab
• 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).
– If problem is MI, LD1 is greater than LD2.
• 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
M. Zaharna Clin. Chem. Lab
Assay for Enzyme activity
• The reaction can proceed in either a forward or
reverse direction
• Lactate + NAD+
LD
LD
Pyruvate + NADH + H+
• The optimal pH:
– for the forward reaction is 8.3 – 8.9
– For the reverse reaction 7.1 – 7.4
Mohammed Laqqan
M. Zaharna Clin. Chem. Lab
• Specimen: Serum, heparin plasma or EDTA plasma
• Measurements & Sources of error
• 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
Clinical Significance
• The measurement of the serum concentration of LD has
proven to be useful in the diagnosis of myocardial
infarction.
• The LD enzyme activity in serum does not rise as much
as CK or AST after myocardial 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