Chapter 2: Diagnostic

Enzymology

E+S ↔ ES → E+P
Learning Objectives for the Chapter

Upon completion of this chapter the student will be

able to:
 Describe the chemical makeup, general
characteristics, classes and nomenclature of
enzymes.
 Discuss how enzymes act as catalysts in
specific biological reactions, in terms of
activation energy and general nature
Learning Objectives for the Chapter

Upon completion of this chapter the student will be

able to:
 Explain plasma specific versus non-plasma specific
enzymes, factors that affect the rate of enzymatic
reactions, including cofactors, coenzymes and inhibitors.
 Describe enzyme kinetics, fixed time assay and
continuous monitoring assay, the unit of enzyme activity
and the calculation for activity and volume activity.
Learning Objectives for Chapter

Upon completion of this chapter the student will be able

to:
• Discuss the biochemical characteristics, source, clinical
significance, methods of analysis, interpretation of
results and sources of errors for selected enzyme tests:
• Transferases
• Phosphatases
• LD, CK
• Amylase and Lipase
Learning Objectives for this
Lesson

Upon completion of this lecture the student will be

able to:
 Describe the chemical makeup and general
characteristics of enzymes.
 Discuss the 6 main classes of enzymes in terms of
general function, listing some common examples.
 Explain the nomenclature for enzymes listing some
common examples.
Learning Objectives for this
Lesson
 Discuss how enzymes act as catalysts in
specific biological reactions.
 Compare activation energy in a catalyzed versus
a non-catalyzed chemical reaction.
 Explain the general relationship between
enzyme, substrate and product; nature of
enzymes in chemical reaction
Learning Objectives for this lesson:

 Explain plasma specific versus non-plasma

specific enzymes
 Describe factors that affect the rate of enzymatic
reactions.
 Discuss the role of cofactors with enzymes
Learning Objectives for this lesson:

 Define enzyme kinetics, fixed time assay and

continuous monitoring assay
 On a Michaelis-Menten curve, identify where a
reaction proceeds in first-order kinetics and
zero-order kinetics
 Recognize a Lineweaver-Burk transformation
and explain why it is useful in describing enzyme
reaction velocity
 Describe three kinds of inhibitors on enzyme
reaction velocity
Learning Objectives

Upon completion of this lecture the student will be

able:

 Compare fixed-time and continuous monitoring

kinetic assays of enzyme activity
 Identify the unit used to report enzyme activity.
 Calculate enzyme activity (volume activity)
Outline

 Diagnostic Enzymology
 Introduction (enzymology from a clinical point of view)
 Classification and Nomenclature of enzymes
 Mechanism of enzymes action
 Nature of enzymes regarding energy requirements of
chemical reaction
 Enzyme kinetics (substrate concentration, temperature,
cofactors, coenzymes, inhibitors, pH)
 Enzyme Assay Techniques
Outline

 Fixed time (fixed time kinetic) assay techniques

 Continuous (kinetic) monitoring assay techniques
 Plasma specific versus non- plasma specific enzymes
 Factors affecting enzyme level in plasma or serum
Outline

 Selected Enzyme Tests

 The transferases (AST, ALT, GGT)
 The phosphatases
 Lactate dehydrogenase
 Creatine kinase
 Amylase
 Lipase
 Principles & techniques for enzyme determination
 Calculation of enzyme activity (volume activity)
 Clinical significance, reporting, documentation and interpretation of
enzyme results
Introduction to Enzymology

 Used for diagnosis and treatment of diseases

 Enzymes are protein catalysts
 Enzymes are present in small quantities in body
fluids
 Enzymes are measured by “what they do”
Enzyme Chemical Makeup

Enzymes are proteins

 Protein structures are composed of :
 Primary bonds
 Secondary bonds
 Tertiary bonds
 Quaternary bonds

 Conjugated with carbohydrates or other

compounds.
Enzyme Characteristics

 Primary structure allows for ionization.

 Tertiary and quaternary structure of enzymes
produces active sites for substrate binding.
Properties of Enzymes

 Temperature dependent activity

 Easily denatured
 Coenzyme and metal activators
Classes of Enzymes:

6 classes of enzymes
1. Oxido-reductase
2. Transferase
3. Hydrolase
4. Lyase
5. Isomerase
6. Ligase
 Name describes type of reaction involved
Nomenclature of Enzymes

 Arbitrary in the past

 Suffix -ase
 Reaction named
 Combination (trivial, common and semi-
systemic)
 Standardized system of names was recognized
Enzyme Nomenclature

 Enzyme Commission of the International Union

of Biochemistry
 unique numerical names consisting of four numbers
separated by periods to indicate class, subclass, sub-
subclass and a specific serial number.
 Lactate dehydrogenase, LD, EC 1.1.1.27
 Alanine transaminase, ALT, formerly serum
glutamate pyruvate transaminase, SGPT, EC
2.6.1.2
Enzyme Nomenclature

 2 Names for each enzyme

 Systematic name: the reactions catalyzed,

associated with a unique numerical code
designation
 Recommended, trivial or practical name: a
simplification , suitable for everyday use.
Mechanism of Enzyme Action

 This equation represents an enzymatic reaction:

 E+S ↔ ES → P+E
 E = enzyme, S = substrate, P = product
 Formation of the ES complex occurs rapidly.

 2 Models for specific binding of substrate to enzyme

 Lock and key specificity (Fisher’s)
 Induced Fit Model
 After binding, enzyme takes a shape complimentary to
substrate
Nature of Enzymes

 Substrate Specificity
 Absolute specific enzymes
 Stereo-isomeric specific enzymes
Nature of Enzymes

 Group specific enzymes

 Bond specific enzymes (function-specific)

Energetics of Catalyzed Chemical
Reactions

Enzymes:
 Act as catalysts in most physiological reactions.
 Lower the activation energy of the substrate
(or reactants) so a reaction can take place.
 Do not change the equilibrium constant of the
reaction.
 Do change rate at which equilibrium is
established.
Catalysts reduce the free or “activation”
energy required to activate a chemical
reaction

Activation energy for

non-catalyzed reaction
Free
energy

Activation energy for

catalyzed reaction

Initial reaction
state
Equilibrium

Reaction
Drawn by John Wentz, MS,CLS
Enzyme Activity

Review Question regarding mechanism of action:

 What is a product?

Answer: compound forming from the

substrate in the chemical reaction.
S+E S-E P + E
Enzyme Activity Review Question:
What is the type of protein that accelerates the
speed of a chemical reaction by binding
specifically to a substrate forming a complex,
lowering the activation energy in the reaction
without becoming consumed or without changing
the equilibrium of the reaction?

Answer: Enzyme
2.1. Introduction to Enzyme Kinetics

 Definition of Enzyme Kinetics:

 The study of the rate of enzyme reactions.

 Factors affecting enzyme kinetics

 Enzyme concentration
 Substrate concentration
 Product concentration
 pH and ionic strength
 Temperature
 Cofactors and Inhibitors
Effects of Enzyme
Concentration on Rate
Ef + S ES  Ef + P

 If substrate concentration exceeds enzyme

concentration, rate is proportional to enzyme activity.
 The basis of clinical assays: excess substrate
available in reagent and unknown concentration of
enzyme in serum.
 ↑ enzyme activity = ↑ rate
Large Amounts of Enzyme
Activity
 When substrate is depleted from a high rate of
product formation, zero order kinetics is no
longer observed.
 Activity needs to be determined by either:
 Diluted sample
 Decreased ratio of sample to reagent
 Fast kinetics

 Final activity is determined by a dilution factor.

Enzyme Activity

 Coupled enzymatic reactions are linked.

 1st enzyme catalyzes a primary reaction
 2nd enzyme catalyzes a secondary reaction
 In vitro coupled reactions:
 secondary enzyme
 provided in the reagent
 produce product
 indicating reaction.
 Secondary enzyme = indicating enzyme
Measuring an Analyte Using
an Enzyme
 Enzymes can be used to measure an analyte
with high level of specificity.
Eg. Ammonia analysis:
NH4+ + 2-oxoglutarate + NADPH
----GLDH ------>glutamate + NADP +H2O
 Only two absorbance readings are taken
 A decrease in absorbance is measured at 340
nm due to the formation of NADP at 37 0C
Effect of Substrate on
Reaction Rate
 Reaction rate increases proportionately with an
increase in substrate concentration, [S].
 Defined as first-order kinetics.

 Km is a constant specific for each enzyme:

the [S] that corresponds to ½ maximum velocity.
 [S] increases until available enzyme is saturated
and reaction velocity flattens out or plateaus.
Rate does not change with added substrate.
 Defined as zero-order kinetics.
Michaelis-Menten Curve
30

Reaction V max = maximum velocity

Velocity (Reaction follows zero-
(v) order kinetics).

15
½ maximum velocity
(Reaction follows first-order
kinetics)

10

10 20
Km ≈ 4
Substrate Concentration = [S]

Drawn by John Wentz, MS, CLS

The Lineweaver-Burk
Transformation
 Determining Vmax using
Michaelis-Menten curve is
1
difficult.
V
 Lineweaver-Burk
transformation is easier
1
because it yields a straight
V max
line plot.
1
 1/v = [Km/Vmax] x 1/[S] +
[S] 1/Vmax
-1
Km
Drawn by John Wentz, MS,CLS
Effect of Product on Reaction Rate

 Accumulated product may inhibit reaction

rate.
Mass action effect
Inhibition
Changes pH
Effect of pH and Ionic Strength on Rate

 Enzymes are proteins.

 Proteins change shape or net molecular charge
as pH changes.
 Most enzymes only work in pH 7.0-8.0
 In-vitro diagnostics (clinical assays) - buffers
used to control pH.
Effect of Temperature
 Chemical reactions rates are increased by increasing
temperature, including enzyme reactions up to optimum
temperature.
 At 40° – 50° C most enzymes are inactivated.
 At 60° – 70° C denatured irreversibly.
 Colder temps.(i.e., 4°C) reversibly inactivate;
storage temp of samples if analysis is to be delayed.
Importance of Temperature

 37°C is ideal for most enzymatic reactions, but

some procedures use 35° or 30° C
 Temperature of rate reactions must be tightly
controlled (± 0.1 ° C). Use of water-bath or other
temperature controlled equipment is necessary.
Some Enzyme Reactions
Require Cofactors (Activators)
 Non-protein cofactors:
Cations: Ca 2+,Fe 2+, Mg 2+, Mn 2+, K +, Zn 2+;
Anions: Cl -, Br –
 Alters enzyme configuration to promote binding
or enable binding site. Increases enzyme
activity.
 Some of these ions are tightly bound to enzyme
molecule, others transiently.
Some Enzyme Reactions Require
Coenzymes

 Coenzymes - class of molecules necessary for

the enzyme to catalyze

 eg. prosthetic group such as NAD/NADP,

vitamins
 Apoenzyme + Coenzyme  Holoenzyme
Inhibitors Interfere with
Enzyme Reactions
 Affect Vmax and Km of enzymatic reactions.
 Three types of inhibitors:
1. Competitive inhibitors.
2. Noncompetitive inhibitors.
3. Uncompetitive inhibitors
Competitive Inhibitors

 Compete with the substrate for the active site of

the enzyme
 prevent formation of product
 have a higher Km than the preferred substrate
 can be overcome by addition of more substrate
 Eg: Lactate and -dehydroxybutyrate for LD
Noncompetitive Inhibitors

 Bind on allosteric site but not the active sites of

enzyme
 Can not be overcome by addition of more
substrate
 Prevents formation of product despite the
enzyme-substrate complex.
Uncompetitive Inhibitors

 Bind to the enzyme-substrate complex

 Prevent the formation of product
 Not overcome by addition of substrate
Enzyme Assay Techniques

2 main types of assay techniques:

 Fixed time kinetic assay techniques
 Continuous (kinetic) monitoring assay
techniques
Fixed-Time (or 2- point) Assays
 Substrate is added and Abs is measured after a
predetermined interval.
 Does not indicate substrate depletion or
presence of inhibitors in reaction system.
 Fixed-time assays are best for batch runs
(multiple samples ran simultaneously)
 If enzyme activity is very high, substrate is
depleted too quickly.
Continuous (kinetic) monitoring assay
techniques
 This is also multi-point
 Abs measurements made at specific intervals
 usually 30 to 60 sec
 Continuous Monitoring refer to a recording
spectrophotometer taking more frequent
measurements
Determining Enzyme Kinetic Activity
Example of Continuous
monitoring: ALT method

Amino acid + substrate –(enzyme,

coenzyme)amino acid + product
Substrate (product of 1st) + coenzyme -(2nd
enzyme) product + reduced coenzyme
 Coupled reaction at 37 0 C
 Decrease in Abs. 340 nm (continuous
monitoring).
Example Enzyme Reaction

L-Alanine + 2-oxoglutarate -- ALT- Glutamate +

Pyruvate
NADH + H+ + Pyruvate -- LDH  Lactate + NAD+
 Absorbance due to NAD can be measured at 340
nm
 The molar absorptivity (epsilon) of NAD at 340 nm
is 6220 cm. L/ mole
 Refer to next slides for results
Enzyme Kinetic Assay

 ΔAbs is determine as Absorbance at time 1

subtracted from Absorbance at time 2
 ΔAbs = A2 – A1
 Sometimes Absorbance decreases with time so
A2- A1 is a negative number.
 International standards have this number indicated
as negative and is multiplied by a negative activity
factor so the final activity is still a positive number.
Example of a Kinetic Assay –
Continued
Time Abs ΔAbs
0 sec .0450
10 .0410 -0.004
20 .0380 -0.003
30 .0330 -0.005
40 .0285 -0.004
Temperature dependent
50 .0255 -0.003
60 .0235 -0.002
ΔAbs = -0.021/min
Decreasing Absorbance Per
Minute
 In ALT the absorbance decreases over time
 The result is -A X F
Min
 Where F340 = -1746
 So final activity is a positive number U/L
 ALT activity would be -0.021 x – 1746 = 37 IU/L
 This results is within the reference range for this
method: 37 IU/L (reference range is 6-37 IU/L)
Plasma specific versus non- plasma
specific enzymes
 Plasma specific enzymes have function in
plasma
 Produced in liver but secreted into plasma
 Clotting factors

 Non-plasma specific enzymes are found in cells

 Produced in specific cells
 Release into plasma during disease
 Amylase, ALT, LD
Factors affecting enzyme level in plasma
or serum
The factors affecting enzyme levels in plasma are:
 Rate of enzyme release from cells
 Extracellular Fluid volume of distribution of the
enzyme
 Enzyme removal rate from plasma (catabolism
or excretion)
 Plasma factors which may affect the method of
assay (inhibitors or activators)
Principles of Enzyme Determination

 Enzyme concentration in serum is not clinically

significant.
 Enzyme recently released from diseased or
dying cells is significant.
 The amount of functional (activity of) enzyme is
significant.
 Enzyme activity is standardized as International
units of activity = IU.
Principle of Enzyme Activity
Determination
 Enzyme activity is measured, since enzyme
concentrations are not clinically significant.
 Some enzyme assays measure the reduction or
oxidate of coenzymes NAD to NADH (or NADH
to NAD) photometrically at 340 nm.
 Enzyme activity is measured when rate is
constant, or zero-order kinetics.
 Temperature, pH, ionic strength must be
maintained.
Enzyme Activity Determination

 Kinetic methods (continuous monitoring)

 Absorbance (Abs) measured at regular intervals
(e.g., 10 or 30 seconds)
 Measurements begin after lag phase
 If there is a fluctuation in temperature, volume,
improper timing, the  absorbance should not be
calculated
 The reaction should be investigated
 The problem should be solved
Enzyme Activity Determination

 Measurements continue until little or no change

in Abs between measurements (substrate
depleted).
 Average change in Abs (Δ Abs)/ minute is
calculated.
Calculating and Reporting Enzyme
Activity/ Volume Activity
 Enzyme activity is reported as International Units/ liter
(IU/L) calculated from  Abs/ min x molar absorptivity
(epsilon) of the product in cm.L/ mol x conversion factor
for volume x ratio of total volume / sample volume in mL.

 Commonly determined as change in  Abs/ min x Factor.

 Review the results again on the next slides
Example of Problems with a
Kinetic Assay
Time Abs ΔAbs
0 sec .0450
60 .0410 -0.004
120 .0380 -0.003
Notice the
180 .0390 +0.001
absorbance readings
240 .0285 -0.0105
are fluctuating here.
300 .0255 -0.003
360 .0235 -0.002
ΔAbs =
International Units of Enzyme
Activity and Volume Activity
 IU = the amount of enzyme needed to convert 1
micromole of substrate to product per minute.
 Volume activity = IU/L
Summary: Enzyme Kinetics
 Enzymes act as catalysts by lowering the
activation energy required for a reaction to take
place.
 The action of enzymes is summarized in the
formula:
E+S ↔ ES → E + P
Question for You:

The equilibrium coefficient (Km) that represents the

likelihood of a particular enzyme-substrate complex to
dissociate and form product is determined from the
Michaelis-Menten curve as_________

Answer: ½ V max
Summary: Enzyme Kinetics –
Continued
 Michaelis-Menten curve describes constant Km,
the substrate conc. that corresponds to ½ V max
– the maximum reaction velocity or rate.
 Lineweaver-Burk transformation gives inverse:
1/Vmax and 1/Km
 But yields a linear line instead of a hyperbolic
curve.
Question for You:

Unexpected low activity results are found for a sample

of known LD activity. The substrate of choice is lactate
but -dehydroxybutyrate is found to be present in the
reagent. When excess lactate is added to the reagent
mix, the observed activity in the known sample is
higher and meets expectations. Adding the additional
lactate describes overcoming _______ inhibition.

Answer: Competitive
Summary: Enzyme Kinetics –
Continued
 The reaction rate increases with substrate
concentration in first-order kinetics. The rate
stabilizes when there is an excess of substrate –
zero-order kinetics.
 Some enzymes require cofactors or activators,
often metallic ions, smaller proteins or vitamins.
Question for You:

The  Abs/min in lactate dehydrogenase analysis was

found to be 0.010 Abs/min. The activity factor (F)
based on molar absorptivity and ratio of sample to
total volume is 4800. What is the LD activity of this
sample in IU/L?

Answer: 0.010 x 4800 = 48 IU/L

Summary: Enzyme Kinetics –
Continued
 Enzyme inhibitors slow or
stop reaction rate. Three
types: Competitive,
Noncompetitive, and
Uncompetitive.
 Enzyme assays are
designed in zero-order
kinetics (constant rate).
Many clinical assays use
fixed-time (end-point)
methodology.
Summary: Enzyme Kinetics –
Continued
 Temperature, pH and ionic strength must
be tightly controlled in enzymatic
reactions.
 Enzymes exist in very small
concentrations in body fluids.
 Enzyme activity is measured and reported.

 The standard unit of enzyme activity: IU/L

References

 Burtis, Carl A., and Ashwood, Edward R.. Tietz:

Fundamentals of Clinical Chemistry. Philadelphia, 2001
 Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. 2007 FA Davis
 Bekele T., Clinical chemistry II lecture notes, Carter
centre.