Transcript Enzymes

Enzymes
Part 1
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:
o without the enzyme becoming consumed
o and without changing the equilibrium of the
reaction.
 A product is produced at the end of the reaction
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Introduction

The catalyzed reactions are frequently specific
and essential to physiologic functions, such as:
o
o
o
o
o
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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. 2015
M. Zaharna Clin. Chem. 2015
General Properties of Enzymes

Like all proteins 1°, 2°, 3°, and 4°
structures
 Active site → cavity where
substrate interacts
o Often water-free site
o Reacts with charged amino acid
residues

Allosteric site
o Another site on enzyme where cofactors or regulatory molecules
interact
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Isoenzymes
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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
o electrophoretic mobility,
o differences in absorption properties
o or by their reaction with a specific antibody
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Cofactors
 Non-protein
molecules required for
enzyme activation
• Inorganic Activators
– Chloride or magnesium ions, etc.
• Organic Coenzymes
– e.g. Nicotinamide adenine dinucleotide (NAD)
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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
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Classes of Enzymes
1. Oxidoreductase:
2. Transferase:
3. Hydrolase:
Classes of Enzymes
4. Lyase:
COO CH2
C-COOCH
+ H2O
Aconitase
COOCH2
C-COOHO C-H
-
COO-
COO
cis-Aconitate
Isocitrate
5. Isomerase:
CH2 OPO3 2Phosphohexose
O
isomerase
OH
OH
HO
OH
a-D- Glucose-6-phosphate
6. Ligase:
ATP + L-tyrosine + t-RNA
CH2 OPO3 2O
H HO
H
H
HO
CH2 OH
OH
a-D-Fructose-6-phosphate
Tyrosine-tRNA
synthetase
L-tyrosyl-tRNA + AMP + PPi
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: cholinesterase, plasmin, thrombin
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Enzyme classification
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.
– An increased plasma concentration of these
enzymes is associated with cell disruption or death
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Factors Affecting Enzyme Levels in
Blood
 Entry
of enzymes into the blood
o Leakage from cells
o Altered production of enzymes
• E.g. increased osteoblastic activity results in
increase in enzymes in bone disease
 Clearance
of enzymes
o Half life vary from few hours to several days
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Factors That Influence Enzymatic
Reactions
o Substrate Concentration
o Enzyme Concentration
o pH
o Temperature
o Cofactors
o Inhibitors
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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
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Measuring enzyme activity

Enzyme activity can be tested by measuring
o Increase of product
o Decrease of substrate
o Decrease of co-enzyme
o 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. 2015
Measuring enzyme activity
 NADH
( a common co-enzyme ) ( the
reduced form ) absorbs light at 340 NM
o NAD does not absorb light at 340 nm
o Increased ( or decreased ) NADH
concentration in a solution will cause the
Absorbance ( A ) to change.
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Measurement Conditions
 Excess
amounts of substrate and any
cofactors or coenzymes
o 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
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Methods for Enzyme
measurement

Fixed time methods
o The reactants are combined,
o The reaction proceeds for a designated time,
o The reaction is stopped (usually by inactivating the
enzyme with a weak acid),
o A measurement is made of the amount of reaction
that has occurred.
o The reaction is assumed to be linear over the reaction
time; the larger the reaction, the more enzyme is
present.
o Possible problems with extremely high enzyme levels
M. Zaharna Clin. Chem. 2015
Methods for Enzyme
measurement
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Continuous-monitoring methods
o Multiple measurements, usually of absorbance
change, are made during the reaction,
o Either at specific time intervals (usually every 30
or 60 seconds)
o or continuously by a continuous-recording
spectrophotometer.
o These assays are advantageous over fixed-time
methods because the linearity of the reaction may
be more adequately verified.
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Methods for Enzyme
measurement
 Continuous-monitoring
methods
o If absorbance is measured at intervals,
several data points are necessary to increase
the accuracy of linearity assessment.
o Continuous measurements are preferred
because any deviation from linearity is readily
observable.
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Measurement Units
 Reported
as “activity” not concentration
o IU = amount of enzyme that will convert 1
μmol of substrate per minute in specified
conditions
o Usually reported in IU per liter (IU / L)
 SI
unit = Katal = mol/sec
o moles of substrate converted per second
o enzyme reported as katals per liter (kat / L)
o 1 IU = 17nkat
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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. 2015
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. 2015
M. Zaharna Clin. Chem. 2015
Creatine Kinase (CK)
– This enzyme is associated with
the regeneration and storage of high
energy phosphate (ATP).
 It catalyzes the following reversible
reaction in the body.
 Action
M. Zaharna Clin. Chem. 2015
Creatine Kinase (CK)

High concentrations of CK in:
o skeletal muscle,
o cardiac muscle
o 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 )
M. Zaharna Clin. Chem. 2015
Creatine Kinase (CK)
 CK
has 3 isoenzymes
 Each isoenzyme is composed of two
different polypeptide chains (M & B)
– CK - BB (CK1)
– CK - MB (CK2)
– CK – MM (CK3)
Brain type
Cardiac type or hybrid type
Muscle type
o Normal serum consists of approximately 94% to
100% CK-MM
o Cardiac muscle CK is 80% CK-MM and 20% CK-MB
M. Zaharna Clin. Chem. 2015
Creatine Kinase (CK)
o BB migrates fastest to anode than MB & MM
o The major isoenzyme in the sera of healthy people is
the MM form.
o Values for the MB isoenzyme range from undetectable
to trace (<6% of total CK).
o It also appears that CK-BB is present in small
quantities in the sera of healthy people
M. Zaharna Clin. Chem. 2015
Diagnostic Significance
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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
o CK-MB increases 4 – 8 hours post AMI
o Peaks at 12 - 24 hours post AMI
o Returns to normal 48 - 72 hours
M. Zaharna Clin. Chem. 2015
M. Zaharna Clin. Chem. 2015
M. Zaharna Clin. Chem. 2015
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. 2015
CK Assay
 Reference
Range for Total CK:
o Male, 15-160 U/L (37°C)
o Female, 15-130 U/L (37°C)
o CK-MB: <6% total CK
M. Zaharna Clin. Chem. 2015
CK isoenzymes

For CK isoenzymes, electrophoresis is the
reference method.
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Other methods include ion-exchange
chromatography, and radioimmunoassay.
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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. 2015
Lactate Dehydrogenase (LD)
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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
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LDH Isoenzymes

Because increased total LDH is relatively nonspecific, LDH isoenzymes can be useful
 5 isoenzymes composed of a cardiac (H) and
muscle ( M ) component

o LDH - 1 ( HHHH )
Cardiac , RBCs
o LDH - 2 ( HHHM )
Cardiac , RBCs
o LDH - 3 ( HHMM )
Lung, spleen, pancreas
o LDH - 4 ( HMMM )
Hepatic
o LDH - 5 ( MMMM )
Skeletal muscle
LD-1 is the fastest towards the anode
M. Zaharna Clin. Chem. 2015
Diagnostic Significance
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LDH is elevated in a variety of disorders.
o
o
o
o
o
in cardiac,
hepatic,
skeletal muscle,
and renal diseases,
as well as in several hematologic and neoplastic
disorders
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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
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Diagnostic Significance

In healthy individuals
o LD-2 is in highest quantity than LD-1, LD-3, LD-4 and
LD-5
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Heart problems: 2-10 X (Upper Limit of Normal)
ULN in acute MI
o If problem is not MI, both LD1 and LD2 rise, with LD2
being greater than LD1
o If problem is MI, LD1 is greater than LD2.
• This is known as a flipped pattern
M. Zaharna Clin. Chem. 2015
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
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Assay for Enzyme activity
• The reaction can proceed in either a
forward or reverse direction
Pyruvate + NAD+
LD
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 : 100-225 U/L (37°C)
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Aspartate Aminotransferase (AST, SGOT, GOT)
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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
M. Zaharna Clin. Chem. 2015
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. 2015
Diagnostic Significance

The clinical use of AST is limited mainly to the
evaluation of hepatocellular disorders and
skeletal muscle involvement.
 Post AMI
o Rises 6 – 8 hours
o Peaks at 24 hours
o Returns to normal by day 5

AST levels are highest in acute hepatocellular
disorders, viral hepatitis, cirrhosis.
o Viral hepatitis may reach 100 x ULN
M. Zaharna Clin. Chem. 2015
Diagnostic Significance

There are two isoenzyme fractions located in
the cell cytoplasm and mitochondria,
o
o
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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. 2015
Assay for Enzyme activity
Measurement by Karmen method – use Malate
dehydrogenase in second step
 Detect change in absorbance at 340 nm

Aspartate + -Ketoglutarate
Oxaloacetate + NADH + H
AST
MD
Oxaloacetate + Glutamate
Malate + NAD
Reference Range : 5 to 30 U/L (37°C)
M. Zaharna Clin. Chem. 2015