CLINICAL ENZYMOLOGY: INTRODUCTION • Inorganic cofactors: Activators : Metalloenzymes

Enzymes o ACTIVATORS
• Are proteins produced by living cells which hasten ▪ alters the spatial configuration of
chemical reactions without altering the equilibrium point enzyme for proper substrate binding
of the reaction or themselves being consumed of changed ▪ chloride, Mg++, Zinc, Ca++, K+
in composition during the process. o METALLOENZYMES =
• Are normally found in all body tissues and appear in serum ▪ Attached to a molecule
following cell injury, degradation of cells or from intact ▪ Catalase, cytochrome oxidase
cells subjected to stress.
• Abnormal large amounts in serum clinically suggests
damage to the organ containing it.
• Are measured in terms of their activity and NOT in terms
of their absolute value.
• catalyze single or limited chemical reactions with a specific
substrate, producing products
KINDS ACCORDING TO location
• unilocular enzymes= found in ONE place ( cytosol)
• bilocular enzymes = found both in cytosol and specific
structure ( mitochondria)
ENZYME STRUCTURE

2+
Mg CPK/ALP
2+
Ca Lipase
Cl-;Br AMS

Active site is often a water-free

cavity, where the substrate A complete and active enzyme system
acts. An allosteric site ( a cavity (holoenzyme) is composed of the
other than the active site) may enzyme portion (apoenzyme) and
bind regulator molecules , thus cofactor
significant to the basic enzyme
structure. When all sites are
filled, no further binding can
occur until an active site
discharges its contents.
• Allosteric enzymes can be
used to regulate enzymatic
pathways. BIOSYNTHESIS OF ENZYME
• controlled by specific genes
COFACTORS • This gives rise to the one-gene=one protein=one enzyme
Are non-protein molecules needed for enzyme activity theory
• Organic cofactors: Coenzymes • each biochemical reaction in the body catalyzed by the
COENZYMES: enzyme is thus controlled by a specific gene
• Known as second substrates • some enzymes need activation . Examples are digestive
• Called prosthetic group when tightly bound to the enzymes that are synthesized from proenzymes or
enzyme zymogens.
• Increasing its concentration will increase the velocity
of an enzymatic reaction
• Essential to achieve absolute enzymatic activity
• Ex. NAD, NADP
Significance of enzyme measurement • the nature of enzyme action that causes the
• It can help monitor the presence and amount of damaged disruption of only certain bonds between atoms
tissue.
• It can help provide clinical information for the diagnosis of
particular diseases or abnormalities.

d. Optical or stereospecificity
• refers to enzymes that combine with only one optical
isomer of a certain compound.
• Ex. Arginase on L-arginine but not on D-arginine
Enzyme activities or concentrations in serum can become elevated
by the following factors
• increase membrane permeability (usually secondary to
cell injury or necrosis)
• increase rates of intracellular synthesis and the
subsequent diffusion of these secretion
into the circulation
SUBSTRATES
SUBSTRATES are reactants to go through chemical changes to yield
products.
ENZYME Specificity
• The structure determines the specificity of enzyme for its
substrate.
Theories on enzyme specificity
a. Emil Fisher’s LOCK & KEY
THEORY: based on the
fitting of substrate (shape
of the key) to a rigid
molecule of enzyme (
shape of the lock).

b. Kochland’s INDUCED FIT

THEORY: change in the
configuration of an
enzyme’s active site ;
Induced by the substrate
• ISOENZYME: enzymes in different forms differentiated
from each other based on certain physical properties
Kinds of specificity such as electrophoretic mobility, solubility or resistance
a. Absolute (substrate) Specificity: to inactivation.
• Acts on one substrate • ISOFORM: enzyme is subject to posttranslational
• catalyzes only a single modifications
reaction. ENZYME CLASSIFICATION/NOMENCLATURE
• ex. Glucukinase acts only on a. Basic types of enzymes
glucose but not on other • food enzymes
hexoses; • digestive enzymes
• Urease on urea • metabolic enzymes
b. Group (relative) Specificity: b. Trivial (arbitrary) name
• an enzyme acts on different substrates falling under a • Also assigned by IUB to shorten systematic names of
particular (functional) group enzymes
• Ex. Pepsin, Trypsin
c. Suffix “ase”
• name of the substrate with the addition of the suffix
“ase”
c. Bond ( linkage) Specificity: • example: enzymes acting on lipids – lipase
enzymes acting on protein – protease
d. Names by Ec of IUB
• 1961: first classification adopted
• 1972;1978: revision of standards assigning a
systematic name to each enzyme.
 • The transition state of ES complex has lower energy of
activation than has the transition state of substrate =
reaction proceeds

Enzyme reaction
• Zero order reaction: reaction rate depends only on
enzyme concentration
• First order reaction: the reaction rate is directly
proportional to substrate concentration

• EC numerical code
• 1st digit: defines enzyme class
• 2nd & 3rd digits: defines subclass and sub-subclass
• 4th digit: defines/indicate the serial number of the
enzyme.
• capital letters are used to name enzymes.
• Changes: GOT-AST; GPT=ALT; CPK=CK; G-6-PDH=G-6-PD;

Factors Influencing Enzyme Reaction

Substrate concentration

a. Michaelis-Menten hypothesis:
“The rate of conversion of
substrate to product is
determined by the substrate
concentration and by the rate of
dissociation of enzyme-substrate
complex”

AMY=AMS
Enzyme kinetics

With the amount of enzyme exceeding the amount of substrate, the

reaction rate steadily increases as more substrate is added.

• In order for a chemical reaction to proceed, the total

energy of the reaction must go down.

• the energy of activation in a chemical reaction is a

measure of energy needed for the conversion of substrate Can enzyme reaction rate increase? However, when the substrate
to the reactive state concentration reaches a maximal value, higher concentration of
• Catalyst is efficient in lowering the energy of activation substrate no longer result in increased rate of reaction (saturation
• E + S ↔ ES P+E kinetics)
WHAT CONCENTRATION SHOULD BE INCREASED? Specimen characteristic
• Hemolysis= mostly increases enzyme concentration
• Lactescense or milky specimen= decreases enzyme
concentration
Nature and Concentration of Products
• the product of E & S interaction can also interfere in the
activity of an enzyme

Enzyme concentration
• an increase in the enzyme concentration produces an
increase in the rate of reaction, provided that all other
factors remain constant but excess amount of substrate is
present.
• The higher the enzyme level, the faster the reaction will
proceed because more enzyme is present to bind with the a. COFACTORS
substrate. • Coenzymes
Temperature • Activators
• an enzyme reaction rate increases as temperature • metalloenzymes
increases. b. Time
• 37 °C= best temperature for most enzymatic reactions • the longer a reaction proceeds, the greater will
• 40-50°C = considered significant. be the concentration of the products.
• 60-65°C =inactivation generally occurs . c. inhibitors
• Q10- 2X INCREASE in reaction rate for every 10°C of • these are known as enzyme poisons.
temperature. • Inhibition can be categorized as noncovalent or
pH= each enzyme has an optimal pH value at which it has a covalent
maximal activity.
• This optimal pH is near the pH of the tissue that contains Types of inhibition
it. a. Covalent inhibition
• Changes in pH can also denature enzyme or influence its • means the enzyme is covalently modified at
ionic state, resulting in structural changes or a change in certain side chains that if they are essential to
the charge on an amino acid residue in the active site. enzymatic activity, would irreversibly inhibit the
Most physiologic pH for enzyme reactions occur between enzyme.
7 to 8 b. Noncovalent inhibition
• occurs when there is a small molecule that
noncovalently attaches to the enzyme-and
changes its conformation or directly prevents
substrate binding.
types of noncovalent inhibitions
1. Competitive inhibition occurs when substrate (S) and
inhibitor (I) both bind to the same site on the enzyme.

ACTIVATOR CONCENTRATION
• an inorganic material that helps bind the substrate to the
active site by forming ionic bridges.
• It also helps orient the substrate so it can attach to the
protein at the proper point and in the correct
configuration
Storage
• -25⁰C= ideal temperature for preservation of enzymes for
• If the inhibitor is bound first to the active site=
longer time
there is no enzyme reaction, no products formed
• 2⁰ to 8⁰C= ideal storage temperature for substrates and
• The inhibition is reversible when the substrate
coenzymes
concentration is significantly increased than the
• 6°C = most enzymes are stable for at least 24 hrs
concentration of the inhibitor.
• 22 ⁰C ⁰C or room temperature= ideal for storage of LD4
and LD5
• -70°C = for CK to retain activity
 2. Noncompetitive inhibition occurs when I binds to both E • Product measurement – starts with zero product level,
and ES. more accurate
• inhibitors bind directly to the enzyme in areas • Coenzyme measurement – measures the increase or
other than the active site decrease in coenzyme
Noncompetitive inhibition maybe Principles of Diagnostic Serum Enzymology
a. Reversible: if only naturally present • Normally, the level of an enzyme in the circulating plasma
metabolic substance bind remains constant
b. Irreversible : inhibitor destroys part of • if the level of that enzyme increases, it could be due to its
the enzyme increased released or decreased removal.
FACTORS AFFECTING SERUM ENZYME LEVEL
Causes on increased serum enzyme levels:
• Impaired removal of enzymes from the plasma
• In pathologic conditions involving tissue necrosis and
degeneration
• Increased permeability of cell membrane
• Physiological or pathological increase in the number of
cells
Causes of decreased enzyme levels
• Increased removal of enzymes from the plasma
• Decreased synthesis due to organ impairment, injury or
3. Uncompetitive inhibition occurs when I binds only to ES
removal
and not to free E.
• Malnutrition leading to decreased enzyme production
• Inhibition occurs
since ESI can not
General methods for enzyme measurement
form product. It
a. Fixed-time
is a dead end
• Reactants are combined
complex which
• Reaction proceeds w/ designated time
has only one fate,
to return to ES. • Reaction stopped
• Increasing the • Measurement
substrate • the reaction is assumed to be linear over the reaction time;
concentration the larger the reaction, the more enzyme is present
results in more ES complexes to which the b. Continuous monitoring
inhibitor binds and thereby increases inhibition. • Also called kinetic assay
Increasing substrate concentration results to • Multiple measurements of changed in absorbance made
increase inhibition. during reaction
4. Mixed inhibition • Preferred than fixed time
• has the ability to bind either the E and ES complex at Kinetic time is advantageous over fixed time methods because the
a different site from the substrate active site linearity of the reaction may be more adequately verified. If
absorbance is measured at intervals, several data points are
necessary to increase the accuracy of linearity assessment.
Continuous measurements are preferred because any deviation from
linearity is readily observable. The most common cause of deviation
from linearity occurs when the enzyme is so elevated that all
substrate is used early in the reaction time.

EXAMPLE OF FIXED TIME

• END-POINT ANALYSIS - the most simple and widely used
technique
Example:
rxn time= 10 minutes

Amount of product = 47.2

Measurement of enzyme activity umol
How are enzymes measured? Rate = 4.72/min for the
• Enzymes are measured in terms of their activity rather volume of sample
than in their concentration. employed
• Catalytic activity: the number of molecules of substance
transformed per minute per catalytic center.
• Molecular activity: the number of molecules of substrate
transformed per minute by one molecule of enzyme
The basis for measuring enzyme activity could be any of the
following:
• Substrate measurement – starts with a high substrate
concentration
Disadvantages of endpoint analysis: Enzymes of clinical importance
• Substrate exhaustion or depletion that occurs when enzyme Clinical significance
sample of high activity is measured
• can’t read the “true” activity because reading is only one Acid phosphatase (ACP) Prostatic carcinoma
point.
• There is no way to know if the reaction has remained Alanine Hepatic disorder
linear all throughout the reaction. aminotransferase (ALT)

Aldolase (ALD) Skeletal muscle disorder

Enzyme As reagents
• to assay the product of one enzyme reaction with the use Alkaline phosphatase Hepatic disorder, bone disorder
of another enzyme reaction. (ALP)
• to quantify substrates to that enzyme
• Enzymes are also used to measure non-enzymatic Amylase (AMY) Acute pancreatitis
constituents of blood like glucose, uric acid, cholesterol.
For enzyme reagents to measure substrate, LDH is an Angiotensin-converting Blood pressure regulation
example. enzyme (ACE)
Immobilized enzymes
Aspartate Myocardial infarction, hepatic
• Chemically bonded to adsorbents like agarose
aminotransferase (AST) disorder, skeletal muscle disorder
• Used for batch analyses, competitive and non competitive
immunoassays Chymotrypsin (CHY) Chronic pancreatitis insufficiency
• More stable than enzymes in solutions
Creatine kinase (CK) Myocardial infarction, skeletal muscle
Enzyme in immunoassay disorder
• Used in competitive and noncompetitive immunoassay for
HIV ab, therapeutic drugs, cancer Ag. Elastase-1 (E1) Chronic pancreatitis insufficiency
• Ex. Horse raddish peroxidase, ALP, G6PD, β-galactosidase
Glucose-6-phosphate Drug-induced hemolytic anemia
• As Indicators= present/absent
dehydrogenase ( G-6-PD)
• Enzymes in these assays function as indicator that reflects
either the presence or absence of analyte. Glutamate Hepatic disorder
Quality Control in Enzyme Assay dehydrogenase (GLD)
• Use of control an available control sera
• Time should be taken into consideration Ƴ-glutamyltransferase Hepatic disorder
• Proper amount of specimen and reagents (GGT)
• Temperature should be strictly monitored Glutathione-S- Hepatic disorder
• Program for Quality Control: transferase (GST)
• adherence to zero order kinetics
• use of pooled frozen serum or stable reference materials Glycogen phosphorylase Acute myocardial infarction
• replicate measurements to evaluate precision of assay (GP)

Lactate dehydrogenase Myocardial infarction , hepatic

Units of Expressing Enzyme Activity
(LDH) disorder, hemolysis, carcinoma
Traditionally enzyme activity is reported in :
• activity units Lipase (LPS) Acute pancretitis
• discoverer’s names ( Bodansky and King units)
• EC standard reporting: 5’-Nucleotidase (5’N) Hepatic disorder
o International Unit (I.U. or U)- equivalent to the
amount of enzyme that catalyzes the conversion Pseudocholinesterase ( Organophosphate poisoning, genetic
of 1 micromole of substrate per minute in an PChE) variants, hepatic disorder,
assay under controlled conditions Suxamethonium sensitivity
• Katal Unit/K.U. ( mol/s)
Pyruvate kinase (PK) Hemolytic anemia
o the SI unit
o equivalent to the amount of enzyme that
Trypsin (TRY) Acute pancreatitis
catalyzes the conversion of 1 mole of substrate
per second under controlled conditions Suxamethonium (succinylcholine)
• Unit recognized by the International System of Units (SI) • Suxamethonium is a depolarizing neuromuscular-blocking
• Enzyme concentration 1 IU=17 nkat drug that consists of two acetylcholine molecules joined
The mol is the unit for substrate concentration and the unit together. At a dose of 1 to 1.5 mg/kg, suxamethonium
of time is second. Enzyme concentration is then expressed causes extremely rapid muscular paralysis, and optimal
as katals/L ( 1.0 IU= 17nkat). When enzymes are intubating conditions are obtained within 30 to 60
quantitated by measuring the increase or decrease of seconds. Can cause hyperkalemia. Suxamethonium is now
NADH @340 nm, the molar absorptivity (6.22 x 103 mol/L) largely replaced by rapid-acting nondepolarising blockers.
of NADH is used to calculate enzyme activity.