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
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
 Enzymes
 are biologic proteins that catalyze (enhance)
biochemical rxns in the biological systems
 all IC & extracellular rxns
 By increasing the rates, results in a more efficient
chemical reaction within a biological system
 The catalyzed reactions are frequently specific &
essential to physiologic functions
 such as the hydration of CO2, nerve conduction, muscle
contraction, nutrient degradation, & energy use.
Introduction to Enzymology
 Found in specific tissue,
 enzymes frequently appear in the serum following cellular
injury or, sometimes, in smaller amounts, from degraded cells
 Enzymology
 is the application of the science of enzymes for the
diagnosis & treatment of disease processes
 Enzymes are
 protein catalysts
 present in small quantities in body fluids
 measured by there catalytic activity or “what they do”
Enzyme Chemical Makeup
 Enzymes are proteins
 are polymers consisting of amino acid units
 As a protein, enzymes have unique structures :
 1º  Amino acid sequence
 2º  Interaction b/n 2 locations on the protein chain
 3º  Folding of chains ( 3D structure )
 4º  2 or more separate polypeptide chains
Enzyme Characteristics
 Primary structure allows for ionization.
 This also influences substrate binding.

 Tertiary & quaternary structure of enzymes produces

active sites for substrate binding.
Enzyme Characteristics….
 Active site
 A pocket or cleft in an enzyme molecule
 Substrates bind to active site making an E-S complex
 After the reaction, the product molecules are released from the active
site.

 Allosteric site
 A cavity other than the active site
 May bind regulator molecules &, thereby, be significant to the
basic enzyme structure
Properties of Enzymes
 Are not consumed in reactions they catalyze;
 but are rather released at the end of the reactions to be
recycled.
 High catalytic efficiency:
 Reaction rates  105 – 1017

 Act under mild conditions (temperature & pressure)

 Easily denatured

 Influenced by coenzymes & metal activators

Properties of Enzymes…

 In addition to the basic enzyme structure,

a nonprotein molecule, called a cofactor, may be necessary
for enzyme activity.

 Inorganic cofactors, such as Cl- or Mg+2 ions are called

activators.

 A coenzyme is an organic cofactor, such as NAD.

Apoenzyme + Cofactor → Haloenzyme

(Catalytically inactive) (active enzyme)
Classes of Enzymes:
 There are 6 classes of enzymes, describing the type of
reaction involved:
1. Oxido-reductase
2. Transferase
3. Hydrolase
4. Lyase
5. Isomerase
6. Ligase
Table-e: Different classes of enzymes and the reactions they catalyze.
Nomenclature of Enzymes

 Arbitrary in the past

 Suffix -ase to the name of S (e.g. urease, amylase)
 Type of reaction involved (eg. lactate dehydrogenase)
 Combination (trivial, common & semi-systemic)
 Standardized system of names was recognized
Enzyme Nomenclature

2 names are given to each enzyme

 Systematic name:
 Chxs. the S acted on, the type of reaction catalyzed (-ase), &,
possibly, the name of any coenzyme involved in the reaction.
 e,.g, L-lactate : NAD+ oxidoreductase

 Recommended, trivial or practical name:

 w/h may be identical with the systematic name but is a
simplification of it, suitable for everyday use
Enzyme Nomenclature
 the IUB system identifies each enzyme by an Enzyme
Commission (EC) numerical code
 containing 4 digits separated by periods to indicate
class, subclass, sub-subclass & a specific serial
number.
E.g.
 Lactatedehydrogenase (LD): EC 1.1.1.27
 Alanine transaminase (ALT /sGPT) : EC 2.6.1.2
Mechanism of Enzyme Action
 This equation represents an enzymatic reaction:
E+S ↔ ES → P+E
E = enzyme, S = substrate, P = product

 The hypothesis of enzyme kinetics assumes the rapid, reversible

formation of ES-complex.
 Transition state

 Active site - contain amino acids that function in S binding,

chemical catalysis, & P release
Models for specific binding of S to
E
1. Fisher’s Lock and key model
 The active site can be assumed to have a rigid structure,
similar to a lock
 Substrate fits in to the active site in the
same manner as the key matches a lock

2. Induced Fit Model

 The flexibility of the protein
 After binding, enzyme undergoes a change in conformation
to fit the substrate
Nature of Enzymes
Substrate specificity
 Depending on which amino acid residues are present on
active sites – S specificity can vary greatly.
 Absolute specific
 Combines with only one S & catalyzes only the one
corresponding reaction.
 Urease acts only on urea to produce ammonia & CO2.
 Stereo-isomeric specific
 Act on one particular stereoisomer of the same S
 Glucose oxidase acts on b-D-glucose only
Nature of Enzymes

 Group specific (function-specific)

 act on all Ss containing a particular chemical group
 Bond specific
 specific to chemical bonds

 E.g; peptidases
Catalytic Mechanism of
Enzymes
 Before changed into P, the S must overcome an "energy
barrier" - activation energy.
 The energy required to raise all molecules in 1 mole of a
compound to the transition state

 The difference b/n the energy levels of the: ground

state & transition state

 To catalyze a chemical rxn  the E forms ES complex,

Energetics of Catalyzed
Chemical Reactions

The enzyme participates in the:

 Making & breaking of bonds, releases the products, &
returns to its original state

E.g; if a S is to be split, a bond might be stretched by the E,

making it to break more easily than in the absence of the E

Enzymes lower the activation energy of the S

(or reactants) so a reaction can take place.
 Activation Energy for Chemical Reactions

Catalysts decrease the amount of energy required to activate chemical

reactions. Enzymes are protein catalysts.
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 Enzyme Kinetics
 Introduction
 Definition of Enzyme Kinetics:
 The study of the rate of enzyme catalyzed reactions.
 i.e., the rate of P formation from S that has bound

specifically to E

Concerned with the:

Quantitative measurement of the rates of E-catalyzed
rxn’s &
Systematic study of factors that affect these rates
Introduction..
 The rate or velocity of a biochemical reaction
 is the change in the concn. of a reactant or product per
unit time.
 A k B
 rate = − d[A] / dt = k[A]

 V0 is determined by the breakdown of ES to form

product
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 [S] exceeds [E], rate is proportional to enzyme activity.

 The basis of clinical assays:

 excess S is available in reagent, & the unknown is the amount of E in
the serum.

 ↑ enzyme activity = ↑ rate

 How quickly the concentrations of reactants & products change
during the reaction
Large Amounts of Enzyme
Activity
 When S 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 - E activity is determined before S
exhaustion occurs
Effect of Substrate on Reaction Rate
 Reaction rate es proportionately with an se in [S].
 Defined as first-order kinetics.

 KM is a constant specific for each enzyme:

 the [S] needed to generate a V0 of ½ Vmax.

 [S] es until available enzyme is saturated & reaction

velocity flattens out or plateaus.
 Rate does not change with added substrate.

 Defined as zero-order kinetics. – [S] >>[E]

Michaelis-Menten Curve
In the presence of a specific enzyme All of the active site of an
30
E are bound by S

V max = maximum velocity

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

15
½ V max
(Reaction follows first-order kinetics)
10
Not all of the active site of
an E are bound by S

Km 10 20
Substrate Concentration = [S]

Effect of [S] on the Vi of an enzyme-catalyzed rxn

Drawn by John Wentz, MS, CLS
Effect of Product on Reaction Rate

 Accumulation of the product of an enzymatic reaction

has an inhibiting effect on the reaction velocity.

 Product inhibition occurs by any of the following ways.

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

 Enzymes are proteins.

 change shape or net molecular charge as pH changes.
 also become charged by accepting or donating H+
depending on the pH environment
 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 ed by increasing T0, including enzyme reactions up to
optimum T0.
 For every 1 degree of T0 raised, E activity ses by 10%
 An es in T0 by 10 °C will double the activity of an E

 At 40 – 50° C most enzymes are inactivated.

 At 60 – 70° C denatured irreversibly.

 Very colder T0 inactivate Es reversibly;

storage T0 of samples if analysis is to be delayed.
Importance of Temperature
 37°C is ideal for most enzymatic reactions, but some procedures
use 25° or 30° C
 Thus, reference ranges for Es need to be based on the T0

 T0 of rate reactions must be tightly controlled (± 0.1 °C).

 Use of water-bath or other T0 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
 When bound tightly to the enzyme molecule, the coenzyme
is called prosthetic group
 eg. prosthetic group - NAD/NADP, vitamins

Apoenzyme + Coenzyme  Holoenzyme

Protein part Non-protein part conjugated active
form of enzyme
Inhibitors Interfere with
Enzyme Reactions
Competitive Inhibitors (CI)
 Structurally resembles S, but is not an S
 Binds to free E at active site where S binds
 Competes with S for free E

 Have a higher Km (Km) than the preferred S

 Can be overcome by sing [S]
 Eg: Lactate and -dehydroxybutyrate for LD
Competitive Inhibitors (CI)…

E.g, The substrate (Succinat) & different competitive

inhibitors of Succinate Dehydrogenase (SD).
Uncompetitive Inhibitors (UCI)
 Is not structurally similar to S
 Binds to the ES-complex
 Not to the active site of the free E

 Prevent the formation of product

 Not overcome by addition of substrate
A  in Vmax & Km
Noncompetitive Inhibitors
 Is not structurally similar to S; is not an S
 Bind on allosteric site but not the active sites of E
 doesNOT compete with S for free E
 Can not be overcome by addition of more S

 Prevents formation of product despite the ES-complex

Enzyme Activity
 B/c Es are usually present in very small quantities in biologic
fluids,
 a convenient method of E quantitation is measurement of
catalytic activity.
 Activity is then related to concentration

 Enzyme activity is a measure of how much enzyme is

present in a reaction.
 The concept of enzyme assays relies on measuring the loss of a
substrate or the increase of a product.
Cont…

 Common methods might photometrically measure

 an increase in product concentration,
a decrease in substrate concentration,
a decrease in coenzyme concentration, or
 an increase in the concentration of an altered
coenzyme.
Enzyme Activity….
 There are two main ways to measure an enzyme's reaction:
Direct or Coupled

Direct
 If the S or P has a characteristic Abs or spectral
"fingerprint," a coloured or fluorescent
 the action of the E itself can be followed by changes in
Abs (e.g. NAD(P)H at 340nm with dehydrogenases),
Enzyme Activity….
 Most enzymes do not produce any change in a readily detectable physical
parameter by their activity.
 This can be overcome using a coupled assays

 After the E under analysis catalyzes its specific reaction,

 A P of that reaction becomes the S on w/h an intermediate auxiliary
enzyme acts.
AP of the intermediate reaction becomes the S for the final reaction, w/h
is catalyzed by an indicator enzyme &
 Commonly involves the conversion of NAD(P) to NAD(P)H or vice

versa.
E.g, the enzyme hexokinase, can be assayed by coupling its
production of glucose-6-phosphate to NADPH production,
using glucose-6-phosphate dehydrogenase (G6PDH).

53
Measuring an Analyte Using an
Enzyme

 Enzymes can be used to measure an analyte with high

level of specificity - Enzymes as reagents
Eg. Glutamate dehydrogenase method for Ammonia analysis:
NH4+ + 2-oxoglutarate + NADPH ------>glutamate + NADP +H2O
GLDH

 Only two absorbance readings are taken

 A decrease in absorbance is measured at 340 nm due to
the formation of NADP at 37 0C
Enzyme Assay

 are laboratory methods for measuring some unique

identifying property of enzymatic activity
 How fast a given amount of S is converted to a P depends
on how much enzyme is present.
 By measuring how much P is formed in a given time,
the amount of enzyme present can be determined

 An assay determines the [P] or [S] at a given time after

starting the reaction.
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

 S is added & Abs is measured after a predetermined

interval, but only 2 readings are taken
 The reaction is assumed to be linear over the entire
reaction time Two
Does not indicate S depletion or presence of inhibitors
in reaction system.
If enzyme activity is very high, S is depleted too
quickly.
 Fixed-time assays are best for batch runs (multiple samples
ran simultaneously)
Continuous (kinetic) monitoring
assay techniques

 also multi-point methods

 Abs measurements are made at specific time intervals
usually 30 or 60 secs., and
 the change observed per unit time is recorded and related
to enzyme activity.
 Measurements are made by a continuous recording
computerized spectrophotometer along the entire reaction
time
Comparison of 2 Point and Multipoint Enzyme
Techniques
Examples of 2-point & Multiple-point Assays

2 - point Assay Multiple -point Assay

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

ALT
L-Alanine + 2-oxoglutarate Glutamate + Pyruvate
LDH
NADH + H+ + Pyruvate 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 Abs at time 1 subtracted from Abs at
time 2
 ΔAbs = A2 – A1

 Sometimes Abs 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
60 .0235 -0.002 Temperature dependent
ΔAbs = -0.021/min
Decreasing Absorbance Per
Minute
 In ALT the absorbance decreases over time
A X F
Min
 Where F
340 = -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)
ENZYMES IN CLINICAL
DIAGNOSIS
Introduction

 Most enzymes are present in cells at much higher

concentrations than in plasma.

 The activity of most enzymes normally detectable in plasma

remains fairly constant in health

 'Normal' plasma enzyme levels reflect the balance b/n

 the rate of synthesis & release into plasma during cell
turnover, &
 the rate of clearance from the circulation.
Plasma specific versus non- plasma
specific enzymes

 Enzymes found in the blood can be classified into 2

 Plasma specific enzymes

 Non-plasma specific enzymes

Plasma-specific enzymes:

 Having a definite & specific function in plasma;

 plasma is their normal site of action.
 Present in plasma at higher levels than most tissues.
 Synthesized by the liver & constantly liberated into plasma
 Become of clinical significance when present in plasma
below normal levels
 as a result of impaired synthesis or an in-born metabolic error.
 e.g, blood coagulation enzymes, the immune response.
Non-plasma-specific enzymes
 Have no known physiologic function in the plasma,
 Present in plasma at much lower concens. than in cells.
 Released into plasma as a result of
 Leakage or cell death in the normal process, or
 Cell damage & death caused by disease.

 Become of diagnostic significance when their plasma levels

increase.
 Secreted enzymes: lipase, amylase, etc,
 Cellular enzymes: amino transferases, LDH etc
Non-plasma-specific enzymes

 Measurement of the non-functional plasma enzyme

levels therefore,
 can provide valuable diagnostic and prognostic clinical
evidence in diseases of the heart, skeletal muscle, liver
and other tissues.
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 distribution of the enzyme
 Enzyme removal rate from plasma (catabolism or
excretion)
 Plasma factors which may affect the method of assay
(inhibitors or activators)
Principle of Enzyme Activity
Determination

 Enzyme concentration in serum is not clinically

significant

 Enzyme activity is significant b/c it relates to

 enzyme recently released from diseased or dying cells.

 Enzyme activity is measured when rate is constant, or

zero-order kinetics.
 TO, pH, ionic strength must be maintained.
Enzyme Activity Determination

 Kinetic methods (continuous monitoring)

 Abs measured at regular intervals (e.g., 10 or 30
seconds)
 Measurements begin after lag phase

 If there is a fluctuation in TO, volume, improper timing,

 the Abs 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 b/n measurements (substrate depleted).

 Average change in Abs (Δ Abs)/ minute is calculated.

Unit of Enzyme activity
 The amount of enzyme in a sample is measured by
 the rate of reaction catalyzed by the enzyme.

 This rate is directly proportioned to the amount of enzyme

& is expressed in enzyme unit, IU/L

 1 IU/L is defined as – the activity of the E w/c transforms

1 μmole of S in to Ps per minute per litter of sample under
optimal conditions
Calculating and Reporting Enzyme
Activity/ Volume Activity

 Enzyme activity is reported as IUs/ liter (IU/L) calculated

from
  Abs/ min x  (of the P in cm.L/ mol) x conversion factor

 Commonly determined as change in

 Abs/ min x Factor.
Example of Problems with a
Kinetic Assay
Time Abs ΔAbs
0 sec .0450
60 .0410 -0.004
120.0380 -0.003
Notice the absorbance
180.0390 +0.001
240.0285 -0.0105
readings are fluctuating
300.0255 -0.003 here.
360 .0235 -0.002
ΔAbs =
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.