Chapter 2: Diagnostic

Enzymology
E+S ↔ ES → E+P
 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
 Definitions
• Continuous Monitoring: A reaction mode in which the
reaction is monitored continuously and the data
presented in either an analog or digital mode.
• Denaturation: The partial or total alteration of the
structure of a protein, without change in covalent
structure, by the action of certain physical procedures
(heating, agitation)or chemical agents.
• Enzyme: A protein molecule that catalyzes chemical
reactions without itself being destroyed or altered.
• First-Order Reaction: A reaction in which the rate of
reaction is proportional to the concentration of reactant.
 Definition
• Fixed-Time Reaction: A two-point reaction mode in which
measurements are taken at specifed times. This mode is
preferred for assays in which the reaction rate is the first
order in regard to the initial substrate concentration.
• Inhibitor: An inhibitor is a substance that diminishes the
rate of a chemical reaction; the process is called
inhibition.
• Isoenzyme: One of a group of related enzymes
catalyzing the same reaction but having different
molecular structures and characterized by varying
physical, biochemical, and immunological properties.
 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- refers to the sequence of amino acid units
that make up the protein strand
– Secondary bonds- refers to the folds of the protein strand.
Sections of the protein strand may be folded into regular
structures,
– Tertiary bonds- refers to folds of sections of the protein
– Quaternary bonds- refers to the association of two or more
peptide strands to form one functioning protein enzyme
• 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.
• Tertiary and quaternary structure of enzymes produces
active sites for substrate binding. Primary structure?
 Properties of Enzymes
• Temperature dependent activity
• Easily denatured
• require Coenzyme and metal activators
• Coenzymes are organic molecules that assist
enzymes in conversion of substrate to product by
contributing H+ or other necessary conditions
• Metal activators contribute to the ionic activity of the
enzyme. Examples, Cl or Mg.
 Classification of Enzymes

1. Oxidoreductases • oxidation/reduction
(eg. Dehydrogenases)
2. Transferases • group transfer
(eg. kinases)
3. Hydrolases • hydrolysis
(eg. proteases)
4. Lyases lysis, • generating double bond
(eg. synthases)
5. Isomerases • rearrangement
(eg. racemases)
6. Ligases • ligation requiring ATP
(eg. synthetases)
 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
act on a group of related substrates rather than
on a single substrate
• Bond specific enzymes (function-specific)
act on substrates containing a particular
functional group or chemical bond
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
 Introduction to Enzyme Kinetics
• Definition of Enzyme Kinetics: quantitative
measurement of the rates of enzyme-catalyzed
reactions and the systematic study of factors
that affect these rates.
• Factors affecting enzyme kinetics
– Enzyme concentration
– Substrate concentration
– Product concentration
– pH and ionic strength
– Temperature
– Cofactors and Inhibitors
 Effects of Enzyme Concentration
Ef + S ES  Ef + P
on Rate
• 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.
 Km
• A constant factor that describes the
relationship between a specific substrate and
how well it binds to an enzyme to create the
optimal amount of product.
• Determined as the amount of substrate
needed to generate a velocity of ½ the
maximum velocity in producing product. Thus
Km = ½ Vmax
• The preferred substrate for an enzyme has the
lowest Km meaning less substrate is needed to
reach a maximum velocity (Vmax) while a
non-preferred substrate has a higher Km.
• The non-preferred substrate needs more
substrate added to reach the maximum
velocity of producing product.
 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/v = [Km/Vmax] x 1/[S] +
1
[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 a-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 absorbance 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
• Absorbance 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 Absorbance 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
50 .0255 -0.003
Temperature dependent
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
q
– 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 D 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 D 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 D 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
180 .0390 +0.001
Notice the
240 .0285 -0.0105
absorbance readings
300 .0255 -0.003
are fluctuating here.
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 concentration 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 a-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