Clinical

Enzymology

ENZYMES
Organic catalyst that hasten a chemical reaction without
themselves being consumed or undergoing a chemical change.
Protein in nature with a high degree of specificity for a certain
substrate or class of substrate
SUBSTRATE
Are substances that are acted upon by enzymes

Enzyme Nomenclature
Enzymes are named acc to the ff:
A. Name of substrate with the addition of the
suffix ase
Ex.
Lipid-lipase
Proteins-protease

 B. Type of reaction they catalyse

Ex.
Transferase
transfer of AMINO GROUP from one substrate to another

Kinase
transfer of PHOSPHATE GROUP from a high energy phosphate
compound to its substrate

Phosphatase
effect of hydrolysis on phosphate esters

Dehydrogenase
removal of hydrogen atoms from its substrate

 C. Numerical designation given by the Enzyme

Commission(E.C.)
Ex.
E.C. 1.1.127 Lactate dehydrogenase
E.C. 3.2.1.1 Amylase

1st numberclass to which the enzyme belongs

2nd and 3rd numbersub class
4thspecific serial number of the enzyme

GENERAL CLASSIFICATION OF ENZYMES

Based on the reaction mechanism each enzyme
catalyzes
1. OXIDO-REDUCTASES
removal or addition of electrons
Reduction-oxidation(Redox) reaction
Oxidation-loss of electron
Reduction-gain of electron

Ex.
Removal of hydrogen Dehydrogenase
Lactate dehydrogenase
Glucose-6-phosphate dehydrogenase

 Lactate dehydrogenase

 2. TRANSFERASES
Catalyze the transfer of a chemical group from
one substrate to another
Ex.
Aminotransferase

-Amino group

Aspartate aminotransferase
Alanine aminotransferase

Kinase/phosphokinase

-Phosphate group

Creatine kinase/creatine phosphokinase

 AST-Aspartate Aminotransferase
SGOT-Serum Glutamate Oxaloacetate Transaminase

 ALT-Alanine Aminotransferase
SGPT-Serum Glutamate Pyruvate Transaminase

 3. HYDROLASES
Hydrolyze the splitting of a bond by the addition of
water(hydrolysis reaction)
Ex.: ALP Alkaline phosphatase

 4. Lyases
Removes groups from substrate without hydrolysis
The product contains double bonds
Ex. Aldolase

 5. ISOMERASES
Intramolecular rearrangement of the substrate compound
Same molecular formula but different physical structure

6. LIGASES
Synthethases
Joins two substrate molecules together

Terms associated with enzymes:

HOLOENZYMES
Active substance formed by the combination of
coenzyme(cofactor) and apoenzyme
HOLOENZYME
APOENZYME
(PROTEIN MOIETY)

COFACTOR
(NON-PROTEIN MOIETY)
COENZYME

ACTIVATOR
VITAMINS
NAD
NADP

METAL IONS
Zn+2
Ca+2

 APOENZYME

Protein portion of an enzyme

Subject to denaturation, in which enzyme loses its activity
Catalytically inactive protein when cofactor is removed
Heat labile and dialyzable

 COFACTOR
Non-protein subs/compounds needed by an enzyme before
enzymatic activity can be manifested
Thermostable and dialyzable
2 types:

A. Coenzyme
B. Activator
A. Coenzyme
Organic molecule
It hasten enzymatic reaction but undergoes a change or is
consumed to another product
Ex.
NAD-Nicotinamide Adenine Dinucleotide
NADP-Nicotinamide Adenine Dinucleotide Phosphate

 B. Activator
Metal ion
In such, the metal ion may serve as:
A. A bridge to hold the substrate and enzyme together
B. The primary catalytic center
C. As stabilizing agent in the conformation for catalytic activity

Ex.
Amylase Cl-, Br
LDHZn+2
Lipase Ca+2
CPK
Mg+2

 ISOENZYME
Enzymes present in an individual with similar enzymatic activity
but differ in their physical biochemical and immunological
characteristics
Ex.
LDH Lactate dehydrogenase

LDH 1
LDH 2
LDH 3
LDH 4
LDH 5

CK Creatine kinase
CK-MB
CK-MM
CK-BB

 METALLOENZYME
Enzyme whose metal ions are intrinsically part of the molecule
such as catalases and cytochrome oxidase

PROENZYME
Inactive precursor of enzymes
Also referred to as zymogen

SUBSTRATE
Substances acted upon by the enzyme which are specific for ach
of heir particular enzymes

ENZYME KINETICS
An enzyme(E) catalyses a reaction by combining with its substrate(S)
to create an enzyme-substrate complex(ES).
E

S ---------ES

The ES complex according to Michelis and Menten can either:

dissociate back to E + S
breakdown to product(P) and free enzyme(provided that the product
has a low affinity for the enzyme)

K2
E

S ---------ES-------- E

K3
E

S ---------ES-------- P

ENZYME SPECIFICITY
Theories that explain the high degree of specificity of an enzyme for
their particular substrate or class of substrate:
1. Emil Fischers LOCK and KEY THEORY:
Rigid enzyme molecule into which the substrate fits
The shape of the key(substrate) must conform into the lock(enzyme)

2. Kochlands INDUCED FIT THEORY

It is based on the attachment of a substrate to the active site of an
enzyme, which then causes conformational changes in the enzyme.
This theory is more acceptable because the protein molecule is
flexible enough to allow conformational changes and also allows
some explanation on the influence of hormones on enzymatic
activity.

TYPES OF REACTION ORDER:

1. Zero Order Reaction
The rate of reaction is linear with the time,
independent of concentration of the substrate and
directly proportional to enzyme concentration.

2. First Order Reaction

The rate of reaction is determined by the
concentration of substrate as well as of enzymes(the
rate of reaction changes continuously with time as the
substrate is consumed.

FACTORS AFFECTING ENZYME REACTIONS:

1. Enzyme concentration
in enzyme concentration, in rate of reaction
2. Substrate concentration
in substrate concentration, in rate of reaction
Upon reaching maximal value of conc. of substrate, it does
not result in increased rate of reaction.
3. Temperature
For every 10 C rise in temp, results to 2-3 times increase in
the rate of reaction
37-40 C optimum temperature for enzyme activity
>40 C-proteins undergo denaturation and loses its
functional ability

 4. Hydrogen ion concentration or pH

some enzymatic reactions proceed at their fastest rate at an
optimum pH.
Extreme pH causes denaturation of enzymes.
Ex.
Pepsin--- active at pH 2.0
Alkaline phosphatase active at pH 8.6-10

Enzyme inhibition:
Occurs when the reaction proceeds at a rate less than
expected due to changes in pH, temperature, substrate and
activator concentration.
Referred to as enzyme poisons

Types of Inhibitor:
1. Competitive Inhibitor
2. Non-competitive Inhibitor

INHIBITORS:
COMPETITIVE INHIBITOR
Substances that compete with the substrate for enzyme binding
because they are chemically analogous to the substrate and bind
to the active sites of the enzyme.
High substrate concentration will overcome the effect of the
inhibitor.

NON-COMPETITIVE INHIBITOR
Substances that does not resemble the substrate and bind to the
enzyme in areas other than the active site.
They do not compete with the substrate.
Increasing the substrate concentration will not overcome the
inhibition.

Units in measuring enzyme

activity
International unit(IU)
Amount of enzyme that catalyzes the conversion of
1 micromole of substrate per minute under controlled condition

Katal unit(KU)
Amount of enzyme that catalyzes the conversion of 1 mole of
substrate per second under controlled condition

Means in measuring enzyme

activity
A. change in coenzyme concentration
B. Increase in product concentration
C. Decrease in substrate concentration
Called an inverse technique

Pitfall in enzyme assays

Hemolysis causes falsely elevated values due to release of
enzymes from RBCs.
Serum rather than plasma is preferred specimen.
Anticoagulants have adverse effect on enzyme activity.

Lactascent or milky serum causes variable absorpton by the

spectrophotometer.
Storage:
Most enzymes are stable at 6 for at least 24 hours.
For a longer period of time, temperature of -20 C or
lower are used to ensure preservation of enzyme activity
CK must be kept at -70 C to retain activity.
For the LD4 and LD5, room temperature only, because it is
inactivated at refrigerator temperature.

Principles of diagnostic serum

enzymology
CAUSES OF INCREASED SERUM ENZYME LEVELS:
Impaired removal of enzymes from plasma.
In pathologic conditions involving tissue necrosis and
degeneration.
Increased permeability of the cell membrane.
Physiological or pathological increase in the number of cells or
increase in the rate of production of cells.

 CAUSES OF DECREASED ENZYME LEVELS:

Increased removal of enzyme from the plasma.
Decreased synthesis due to organ impairment, injury or removal.
Malnutrition leading to decreased enzyme production.

oxidoreductases
A. LDH
E.C.: 1.1.1.27
Recommended Name: Lactate dehydrogenase
Systematic name: L-Lactate: NAD+ oxidoreductase

Action:
Conversion of lactic and pyruvic acid
Hydrogen-transfer enzyme
Coenzyme: NAD

 EQUATION:

 Tissue source:
Heart, liver, skeletal muscle, kidney and RBCs
Lungs, smooth muscles, brain

Diagnostic Significance:
Cardiac, hepatic, skeletal muscle, renal and hematologic dse.
Highest level is seen in pernicious anemia and Hematologic
disorders
In AMI, LDH rise within 12-24 hrs
peak within 48-72 hrs
remains elevated for 10 days

Ldh isoenzymes
ISOENZYME
14-26%

TISSUE

DISORDERS:

Heart
RBC

MI
Hemolytic anemia

LDH-1
(heat
stable)

HHHH

LDH-2
(heat
stable)

HHHM 29-39%

Heart
RBC
Renal cortex

Megaloblastic anemia
Acute renal infarction
hemolysis

LDH-3

HHM
M

20-26%

Lung
Lymphocytes
Spleen
Pancreas
Brain

Embolism
Pneumonia
Lymphocytosis
Pancreatitis
CA

LDH-4
HMM
(heat labile) M

8-16%

Liver

Hepatic injury

LDH-5
MMM
(heat labile) M

6-16%

Skeletal muscle

Skeletal muscle injury

 LDH-6
Alcohol dehydrogenase
Increased in Arterosclerotic cardiovascular failure
Elevated level signifies grave prognosis and impeding death
Techniques in measuring LD isoenzymes:
A. Physical
Electrophoresis
Selective absorption on Diethylaminoethyl cellulose(DEAE)
Solvent precipitation technique
Heat denaturation at 65 C for 30 mins

B. Chemical
Substrate-product relationship
Coenzyme affinty

Differential inhibition of LD activity

C. Immunological test

 Sources of error:

Hemolysis
Unstable serum
Spx should be stored at 25 C and analyzed within 48 hrs
LDH-5 most labile

Clinical Significance:
LD levels are markedly increased in the ff:
Megaoblastic anemia
Pulmunary infarction
Granulocytic leukemia Hodgkins Dse
Hemolytic anemia
Infectious mnonucleosis
Progressive Muscular Dystrophy(PMD)

Glucose-6-phosphate
dehydrogenase
E.C.: 1.1.1.49
Recommended name: Glucose-6-Phosphate Dehydrogenase
Systematic name: D-glucose-6-phosphate: NADP+ 1oxidoreductase

Action: oxidation of glucose-6-phosphate to 6phosphogluconate

 Tissue Source:
Adrenal cortex, spleen, thymus, lymph nodes, lactating
mammary glands and RBCs
Diagnostic Significance:
RBC
NADPH is converted into glutathione

Glutathione
Anti-oxidant
protects hgb from oxidation
Dec. G6PD=dec. NADPH=dec. Gluthathione

Dec. Gluthathionemay cause hemolysis/damage to cell membrane

 Decreased G6PD
G6PD deficiency
Inherited sex-linked trait
May cause drug-induced hemolytic anemia/exposure to oxidizing
substances

Increased G6PD
Myocardial Infarction and Megaloblastic anemias

Assays:
Specimen used:
Deficiency of enzyme red cell hemolysate
Elevation of enzyme level serum

Reference Range:
10-15 u/g hgb

TRANSFERASE

AST
E.C.: 2.6.1.1
Recommended name: Aspartate aminotransferase
Systematic name: L-aspartate:2-oxaloglutarate
aminotransferase

Action:
Transaminase-synthesis and degradation of amino acid
Transfer of amino group between aspartate and aketoglutarate

 Tissue source:
Cardiac muscle, liver, skeletal muscle
Kidney, pancreas, RBC

Diagnostic Significance:
Hepatocellular and Skeletal Dse.
AMI
AST rise at 6-8 hrs
Peak at 24 hrs
Return to normal within 5 days
Decreased in
pregnant women

Assays:
Rietmann-Frankel Method
Substrate: aspartic alpha ketoglurate
Color developer: 2,4 dinitrophenyl hydrazine
End Product: glutamic acid and oxaloacetic acid
End color: brown

Karmen Method
Indicator: Malate dehydrogenase
Absorbance: 340 nm
pH: 7.3-7.8

Babson and Read Method

Color developer: diazonium salt
End color: violet

Source of error:

Hemolysis

Falsely increased results

Alcohol lowers AST values
Muscle trauma like intramuscular injectiions, exercise, or surgical operation can significantly increase ATS levels

Reference Range: 5-30 u/L

ALT
E.C.: 2.6.1.2
Recommended name: Alanine aminotransferase
Systematic name: L-alanine: pyruvate aminotransferase

 Tissue source:
Liver
Liver-specific enzyme
Diagnostic significxanc:
Hepatic Disorders

Assay:
Reitman and Frankel Method
Substrate: alanine alpha ketoglutarate
End Product: glutamate and pyruvic acid

Walker method

 Assay:
Indicator: LDH
Absorbance:340 nm
pH: 7.3-7.8
Source of error:
Stable for 3-4 days at 4 C
Reference
range: 6-37 u/L

 CK
E.C.: 2.7.3.2
Recommended name: Creatine kinase, creatine
phosphokinase
Systematic name: ATP: Creatine N-phosphotransferase
Action:
Associated with ATP regeneration in contractile and transport
systems
Physiologic Function:
Muscle cells, creatine phosphate
Production of ATP

 Tissue source:
Skeletal muscle tissue, Heart muscle, brain
Bladder, placenta, GI tract, thyroid, uterus, lungs, pancreas,
prostate and spleen

Diagnostic Significance:
Disorders of the Heart, and Skeletal muscle tissue
Sensitive indicator of AMI and muscular dystrophy

Ck isoenzymes
ISOENZYME

LOCATION

CK-1

CK-BB(BRAIN)

BRAIN, BLADDER, LUNG,

PROSTATE, UTERUS,
COLON, STOMACH,
THYROID

CK-2

CK-MB(HYBRID)

CARDIAC, SKELETAL
MUSCLE

CK-3

CK-MM(MUSCLE)

CARDIAC, SKELETAL
MUSCLE

CK-BB
Highest concentrations in CNS, GI tract and uterus
Elevated in px with CA
Useful tumor-associated marker
Increased in CNS damage, tumors, child-birth

CK-MB
Major isoenzyme found in striated muscle and normal serum
Elevated in cardiac disorders
In AMI, CK rise at 4-8 hours
Peak in 12-24 hrs
Return to normal within 48-72 hours

 Macro-CK
Midway between CK-MM and CK-MB
CK-Mi(mitochondrial CK)
muscle, brain, liver

Assay:
Electrophoresis
Reference method used for measurement of isoenzymes
Ion-exchange Chromatography
Radio Immunoassay(RIA)
Immuno-inhibition

 Reference range:
Total CK:
Male: 15-60 u/L
Female: 15-130 u/L

GGT
E.C.: 2.3.2.2
Recommended name: Gamma-glutamyl transferase
Systematic name: (5-glutamyl) peptide: amno acid-5-glutamyl
transferase

Action: transfer of gamma-glutamyl residue from gammaglutamyl peptides to amino acid, H2O and other small
peptides
Involved in peptide and protein synthesis, regulation of tissue
gluthathione levels and transport of amino acid across cell
membrane

 Tissue source:
Kidney, brain, prostate, pancreas and liver

Diagnostic significance
Liver
Biliary ductules
Hepatobiliary disorders
Increased in patients taking drugs such as warfarin, phenobarbital
and phenytoin
Alcoholism
GGT assays:
Useful in monitoring the effects of abstention from alcohol and are
used by alcohol treatment centers

 Assay:
Substrate: gamma glutamyl-p-nitroanilide
Absorbance: 405 to 420 nm
Source of error:
Stable for 1 week at 4 C
Reference Range:
Male: 6-45 u/L
Female: 5-30 u/L

Hydrolases
I. Phosphatases
Characterized by the ability to hydrolyze a large variety of organic
phosphate esters with the formation of an alcohol and a phophate
ion.
There are two main types:
1. Alkaline Phosphatase(ALP)
2. Acid Phophatase(ACP)

1. ALP
E.C.: 3.1.3.1
Recommended name: Alkaline phosphatase
Systematic name: orthophosphoric monoester phosphorylhydrolase

Action:
Hydrolysis of various phophomonoesters at alkaline pH
Liberate inorganic phosphate from an organic phosphate ester with
production of an alcohol

 Optimum pH: 9.0-10.0

Activator: Mg 2+
Tissue source:
Intestines, liver, bone, spleen, placenta, kidney, RBC

Diagnostic Significance:
Increased in:

Hepatobiliary and Bone disorders

Obstructive conditions
Pagets dse, Osteomalacia, Ricketts
Pregnancy

Decreased in:
Hypophosphatasia
Malnutrition

 Considerations in ALP assays:

Physiologically elevated in growing children and in pregnant
women in the third trimester
EDTA inactivates ALP owing to chelation of magnesium ions

Alp isoenzymes
ISOENZYME
LIVER

FASTEST

BONE

HEAT LABILE

PLACENTA

HEAT STABLE
INHIBITED BY PHENYLALANINE

INTESTINES

SLOWEST

 Regan-Nagao Isoenzyme
Carcino-placental ALP
Detected in various CA(lung, breast, ovarian and colon)

Assay:

 Assays:
A. Substrate: beta-glycerophosphate
End Product: inorganic phosphate+glycerol
1. Bodansky
2. Shinowara
3. Jones
4. Reinhart
B. Substrate: phenylphosphae
End product: phenol
1. King and Armstrong

 C. Substrate: p-nitrophenyl phosphate

Product: p- nitrphenol or yellow phenoxide ion
1. Bessey, Lowry and Brock
2. Bowers and Mc comb
D. Substrate: phenolphthalein diphosphate
Product: phenolphthalein red
1. Huggins and Talalay
E. Substrate: alpha naphthol phosphate
Product: alpha naphtol
F. Substrate: Buffered phenolphthalein phosphate
End product: free phenolphthalein
1. Klein, Babson and Read

 Klein, Babson and Read

Substrate: Buffered phenolphthalein phosphate
End product: free phenolphthalein

Color developer: NaOH

End color: pink
Bessey, Lowry and Brock
Substrate: p-nitrophenyl phosphate
Product: p- nitrphenol or yellow phenoxide ion
Color developer: 0.02 N NaOH
End color: yellow

ACP
E.C. : 3.1.3.2
Recommended name: Acid phosphatase
Systematic name:

 Tissue source:
Prostategland
RBC, Platelet, liver, spleen, milk, bone marrow
To differentiate Prostatic ACP and RBC ACP inhibitors are
added:
L-tartrate ions; Prostatic ACP
Formaldehyde and Cupric ions: RBC and Platelet ACP

Total ACP-ACP after the addition of L-tartrate= Prostatic ACP

Optimum pH: 4.9-5.0

 Assays:
Methods:

Substrate

End product

1. Bodansky

Beta-glycerophosphate

Inorganic phosphate

2. Gutman and Gutman

Phenyl phosphate

phenol

3. Fishman and Lerner

Phenyl phosphate

phenol

4. King Armstrong

PNPP

P-nitrophenol

5. Shinowara

PNPP

6. Bessy, Lowry annd

Brock

PNPP

7. Babson, Read and

Phillips

Alpha naphthyl
phosphate

Alpha-napthol

8. Roy and Hillman

Thymolphthalein
monophosphate

Free thymolphthalein

 Roy and Hillman

Thymolphthalein monohosphate
Most specific substrate for ACP
Color developer: Na OH- Na Carbonate solution
End color: blue
Babson, Read and Phillips
Color developer: tetra azotized orthodianisidine
End color: brown

 Source of error:
Serum ACP is higher than plasma ACP
Hemolysis
Diagnostic significance:
Increased in:
Metastatic prostate carcinoma

A. moderate elevation of TOTAL ACP

Female breast CA
Pagets Dse
Hyper parathyroidism

B. Non-Prostatic ACP elevation

Niemann-Picks dse
Gauchers dse
Myelocytic leukemia

Also present in seminal fluid

Used in forensic cases such as in rape

 Reference range:
Prostatic ACP
0-35 IU/L

Total ACP
Male: 2.5-117 IU/L
Female: 0.2-3.5 IU/L

AMS
E.C. : 3.2.1.1
Recommended name: a-amylase
Systematic name: 1,4-D-glucan glcanohydrolase

Action:
Breakdown of starch and glycogen

2 isoenzymes:
1. Salivary
Ptyalin(fast)
2. Pancreatic
Amylopsin(slow)

Starch/glycogen glucose, maltose, dextrins

Activators:
Ca2+
Cl-

 Tissue source:
Pancreas, salivary glands
Skeletal muscles, small intestine, fallopian tube
Diagnostic Significance:
Acute pancreatitis
Levels reaches 4-6 times the normal value and normalizes
within 3-4 days
Mumps, perforated petic ulcers, appendicitis, ruptured ectopic
pregnancy, dissecting aortic aneurysm, biliary tract disease

 Assay for enzyme activity:

Amyloclastic
Decrease in substrate concentration/disappearance of starch
Saccharogenic
Amount of reducing sugars produced
Chromogenic
Measures increasing color from production of product coupled with
chromogenic dye
Continous monitoring
Coupling of several enzyme system to monitor amylase activity

Reference Range:
Serum:
25-130IU/L
Urine
1-15 IU/L

Considerations in AMS assays:

Mouth pipetting is not done
Lipemic specimens cause a reduction in amylase activity

LPS
E.C. : 3.1.1.3
Recommended name: Lipase
Systematic name: Triacyl glycerol acylhydrolase

Action:
Hydrolisis of esters in the alpha-position to yield betamonoglycerides and fatty acids

 Tissue Source:
Pancreas, stomach and small intestine
Diagnostic Significance:
Acute pancreatitis

Assay for enzyme Activity:

Cherry-Crandal
Substrate: olive oil
Indicator: phenolphthalein
End color: pink
Incubation period: 24 hrs

Tietz and Templaton

Indicator: Thymolphthalein
End coor: blue
Incubation period: 4-6 hrs

Source of error:
Hemolysis
Falsely decreased result
Hgb acts as inhibitor for enzymatic activity

Reference Range:
0-1.0 IU/mL
1.0-1.5 cherry-crandal unit

lyases

ALDOLASE
E.C.: 4.1.2.13
Recommended name: Fructose diphophate
Systematic name: D-D-fructose-1, 6-bidiphosphate, Dglyceraldehyde phosphate lyase
Action:
Spitting of D-fructose di phosphate to D-glyceraldehyde
phosphate and dihydroxy acetone phosphate

3 isoenzymes:
A. Aldolase AB. Aldolase BC. Aldolase C-

skeletal muscle
liver, kidney and WBC
brain

Assay:
Pinto, Kaplan and Van Dreal
Sibley and Lehninger

 Diagnostic Significance:
Severe:
Muscle degradation
Viral hepatitis

Moderate:

Gangrene
Megaloblastic anemia
Granulocytic leukemia
Metastatic liver CA
Psychosis
Trichinosis