Enzymology

Professor Ann Simpson

School of Life Sciences

For each group of enzymes, you should take special
note of:
1. The reaction in the body that the enzyme catalyses
2. If appropriate the number and variety of enzyme
forms.
3. The clinical significance of the enzyme
4. The method of measurement in the Clinical Lab

Enzymes are present in all body cells, and each one

functions in a specific reaction. Enzymes catalyse all
essential reactions, such as oxidation, reduction,
hydrolysis and synthesis, that supply energy and
chemical changes necessary for vital activities of your
body, like muscle contraction, respiration, maintenance
of body temperature and so forth. As catalysts,
enzymes are not used up in the process and do not
change the equilibrium point of the reaction; they
merely accelerate the rate for reaching the reaction.
Factors Governing the Rate of Enzyme-
Catalyzed Reactions
1. Temperature
2. pH
3. Concentration of Enzyme and Substrate
 Most enzyme kinetics can be
summarised by the Michaelis-Menten
Model
 Enzymes
Enzymes are present in all body cells (intracellular) and body fluids
(extracellular)
Each enzyme functions in a specific reaction
Enzymes catalyse all essential reactions such as:
oxidation
reduction
hydrolysis and synthesis
Supply energy and chemical changes necessary for vital
activities of your body like:
muscle contraction,
respiration
maintenance of body temperature
As catalysts are reusable: enzymes are not used up in the process and do
not change the equilibrium point of the reaction
"they merely accelerate the rate for reaching the reaction.
Enzyme Classification
 Enzymology- 2 Major Groups
Digestive - Digestive enzymes
are secreted along the entire
gastrointestinal tract and
break down food into particles
that can be readily absorbed
and assimilated by the body
Metabolic- Metabolic enzymes
are produced internally,
responsible for running the
body at the blood, tissue and
organ level.Needed for: new
cell growth, repair and
maintenance of all bodys
organs andfunctions
 CLINICAL APPLICATIONS OF
ENZYMES.
Enzyme assay in the diagnosis of disease is one of the
most frequent clinical laboratory procedures. The most
commonly used fluid for this purpose is serum, the fluid
that appears after the blood has clotted. The liquid portion
of unclotted blood is called plasma. Serum is used for
many enzyme assays because the preparation of plasma
requires addition of anticoagulants that interfere with
some assays.
Enzymes in circulating plasma are either plasma specific
or non-plasma specific.
 Plasma specific enzymes
Enzymes that are normally present in plasma, perform
their primary function in blood, and have levels of
activity that are usually higher in plasma than in tissue
cells
eg. enzymes involved in blood clotting (thrombin), fibrinolysis
(plasmin), complement activation (cholinesterase).
These enzymes are mainly synthesised in the liver and
released into the circulation at a rate that maintains
optimal steady-state concentrations. Hereditary
enzyme defects or impaired liver function can cause
suboptimal levels.
eg. deficiency of thrombin can cause defective blood
coagulation or fibrinolysis.
Non-plasma specific enzymes .
Intracellular enzymes are normally present in
plasma at minimal levels or at concentrations
well below those in tissue cells, but are
released into the body fluids in excessive
concentrations as a result of cellular damage
or impairment of membrane function.
Distribution of Diagnostically Important Enzymes
Enzyme Principle Sources
Acid Phosphatase Prostrate, erythrocytes
Alanine Liver, skeletal muscle, heart
aminotransferase
Alkaline Phosphatase Liver, bone, intestinal mucosa,
placenta, kidneys
Amylase Salivary glands, pancreas,
ovaries
Aspartate Liver, skeletal muscle, heart,
aminotransferase kidney, ethrocytes
Cholinesterase Liver
Creatine kinase Heart, skeletal muscle, brain,
smooth muscle
-Glutamyltransferase Liver, kidney
Lactate Dehydrogenase Heart, liver, skeletal muscle,
erythrocytes, platelets, lymph
nodes
Principal Clinical
Applications
Carcinoma of prostrate
Hepatic parenchymal disease
Bone disease, hepatobilary
diseases
Pancreatic diseases
Myocardial infarction, hepatic
parenchymal disease, muscle
disease
insecticide poisoning,
suxamethonium sensitivity,
hepatic parenchymal disease
Myocardial infarction, muscle
diseases
Hepatobilary disease,
alcoholism
Myocardial infarction,
haemolysis, hepatic
parenchymal diseases
Factors Affecting Enzyme Levels
in Plasma or Serum
Lipid metabolism :
Conversion of excess carbohydrate
into triglyceride.
Packaging of lipids from meals
(chylomicrons) into lipoproteins
(eg., VLDL)
Cholesterol synthesis.
 Lipid Metabolism

Conversion of
carbohydrate into
triglyceride (bad
cholesterol)

Packaging if lipids
from meals
(chylmicrons) into
liporproteins (eg.
VLDL)
 Design of an enzyme assay
The key element is to select a suitable chemical or
physical technique for following the appearance
of a product or the disappearance of a substrate.
if the reaction is accompanied by a change in optical
properties it can be monitored continuously by
photometric procedures such as light absorption or
fluorescence.
Two Step Procedures:
In assaying many enzyme systems a two- step
assay procedure is the easiest to perform
 ISOENZYMES

Isoenzymes or isozymes are multiple forms of an

enzyme family that catalyse the same biochemical
reaction. They usually however share the same
molecular weight, but may differ in the following
properties-
1. Each isoenzyme of a family has a different affinity for
substrates and cofactors. Thus the Michaelis
constant (Km) and specificity for different
substrates may vary.
2. The various isoenzymes of a family may differ in the
ability to be inhibited by specific agents.
3. They may differ in their physical properties, such as
heat stability, charge, and amino acid composition.
 Lactate Dehydrogenase
LD is an essential enzyme in the Emden-Meyerhof
glycolytic pathway for the utilisation of glucose. It
permits organisms to generate a temporary oxygen
debit by accumulating lactate, which is oxidised to
pyruvate when oxygen becomes available.
The tissue levels of LD are about 500-fold higher
than the level in serum, therefore even minor tissue
damage can lead to a detectable increase of total LD
in the blood. The specific isolation of LD isoenzymes
is therefore very useful in pinpointing the location of
tissue damage.
There are a total of five isoenzymatic forms,
designated LD1, LD2, LD3, LD4 & LD5 normally
detected in several tissues including the heart,
muscle and liver.
Subunit Structure and Tissue Distribution of LD-
isoenzymes

Isoenzyme Form Subunit Structure Major Organs (Tissues)

LD1 (HHHH) H4 Heart, erythrocytes, pancreas renal cortex

LD2 (MHHH) H3M Heart, erythrocytes, lungs, thyroid, spleen, brain,

lymph

LD3 (MMHH) H2M Lung, lymph nodes, spleen, adrenals, thyroid, brain

LD4 (MMMH) HM3 Skeletal muscle, lungs, adrenals

LD5 (MMMM) M4 Liver, granulocytes, skeletal muscle

 Methods of LDH Measurement

1. Electrophoresis on cellulose acetate and

agarose gels.
2. Substrate specificity method.
3. Immunoprecipitation.
 SUBSTRATE SPECIFICITY
Advantages of Lactate to Pyruvate Reaction;
1. Substrate inhibition by lactate is less than that produced
by pyruvate.
2. NAD+ preparations appear to contain fewer
endogenous LDH inhibitors than NADH preparations
used in the reverse assay.
3. The reaction linearity of this reaction is more
prolonged.

Advantages of the Pyruvate to Lactate Reaction:

1. Less expensive assay because of much lower
concentration of reagents.
2. Greater change in absorbance with time.
3. Greater stability of working reagents once they are
prepared.
IMMUNOPRECIPITATION
 IMMUNOPRECIPITATION
TECHNIQUE
In this technique a goat Ab against the M
subunit reacts first against the M subunit-
bearing LD2-5 in the patients serum. An
anti-goat immunoglobulin conjugated with
polyvinyl fluoride is then added to precipitate
the goat Ab-LD isoenzyme complex. After
the precipitate has been separated, the LD
activity of the supernatant is measured; the
result represents the quantity of LD1.
 SPECIMEN REQUIREMENTS
Serum is generally used for total LD and isoenzyme studies,
and samples can be stored at room temperature for 2-3
days without loss of activity. Since LD4 and LD5 are
sensitive to the cold, refrigeration of the specimen is not
advisable unless NAD+ or glutathione is added to retard
the rate of inactivation of these two isoenzymes.

The following types of specimens should be avoided:

1. Haemolysed specimens, as erythrocytes contain a high
concentration of LD1 and 150X more LD than serum.
2. Plasma- LD is 4X higher in serum than plasma owing to
the release of LD3 from platelets during blood clotting.
3. Anticoagulants that contain citrate, oxalate and fluoride
inhibit LD.
TIME COURSE OF PLASMA ENZYME
CHANGES AFTER MYOCARDIAL
INFARCTION
 TIME COURSE OF PLASMA ENZYME
CHANGES AFTER MYOCARDIAL INFARCTION
 LD 6
Recently, about 40 odd observations of an LD6
isoenzyme have been reported in the literature. This
enzyme is cathodal to LD5, and has been seen in
patients with several conditions. The common
features of these cases was that all patients had severe
liver disease or circulatory failure. Although the
origin of this isoenzyme is as yet unknown, it is
definitely released from damaged liver.

 LDH ISOENZYME IN DISEASE

Liver Diseases: although the liver contains
more LD than cardiac muscle, a significant
change in LD levels is relatively uncommon in
liver disease. This is probably related to the
short half-life of the major liver isoenzyme.
However, when LD5 isoenzyme instead of
total LD is measured, it becomes a very
sensitive marker for liver damage including
hepatitis, intoxication, circulatory disturbance
and neoplasm.
 LDH ISOENZYME IN DISEASE
Skeletal Muscle Diseases:
Normal skeletal muscle contains mostly LD5, and in
many inflammatory diseases of the muscle, including
trauma LD5 is markedly increased. Even strenuous
exercise may cause transient elevation. However, for
diagnosis of muscular diseases, CK and aldolase are
more sensitive and should be carried out when an LD
isoenzyme pattern is suggestive of myopathy.
Malignancy: in 41% of patients with malignant tumours,
total Ld is elevated. Elevation is usually seen in the
late stage of malignancy or when metastasis has
taken place. In most tumours , LD4 and LD5 are
predominant because of the anaerobic metabolism of
the neoplasm.
.
CREATINE KINASE
 CREATINE KINASE
CK is a dimer with a molecular mass of approx. 80,000
daltons. Its basic subunits as I mentioned before are
CK-M and CK-B, and by various combinations three
isoenzymes- CK-MM, CK-MB, CK-BB are formed. In a
similar fashion to the LD isoenzymes these enzymes
are named for their tissue of origin, therefore CK-MM
has the highest concentration in the skeletal muscle
and CK-BB in the brain, while CK-MB is a hybrid of 2
subunits and is found mostly in the heart. In addition to
these 3 cytoplasmic isoenzymes , there is a
mitochondrial isoenzyme designated CK-m. It has
been found in heart muscle, skeletal muscle , brain and
liver, but it is not present in serum under normal
conditions.
 Methods of CK Measurement
1. Electrophoresis
2. Immunological Methods
ELECTROPHORESIS
 IMMUNOLOGICAL METHODS
For measuring CK isoenzymes require specific antisera
against M & B subunits. These are obtained by preparing
monoclonal or polyclonal antibodies. These methods
include RIA's & immunoinhibition techniques which both
tend to lack specificity and therefore inadequate for clinical
labs. Current practice employs the "sandwich"
immunoenzymatic technique, in which the B subunits of
CK-MB and CK-BB if present are bound by anti-CKb sera
rendered immobile on a matrix, such as a bead,, and then
any M subunits associated with these bound B subunits,
such as CK-MB are estimated with the use of an anti-CK-M
Ab. This AB may be either labelled with a radioactive tracer
or conjugated with alkaline phosphatase or peroxidase, the
activity of this enzyme can be measured and is proportional
to the CK-MB concentration in the serum.
 SPECIMEN REQUIREMENTS.
Serum is the specimen of choice. If plasma
is used, heparin is the anticoagulant of
choice.
EDTA, citrate and fluoride can inhibit CK
activity.
Ck is stable for 1 day at RT, 7 days at 4oC
and 1 month at -20oC.
CK is unstable in light
 CLINICAL SIGNIFICANCE OF CK
ISOENZYMES
Myocardial Infarction- this is the major clinical
application of CK isoenzymes. The determination of
CK-MB is still the most reliable lab method for
establishing the presence of AMI. Serial blood
samples should be drawn every 8 to 12 hrs after the
patients admission to detect the peak and subsequent
fall of CK and Ck-MB. CK-MB rises 7-8 hrs after onset
of symptoms, peaks at 18 hrs and returns to normal
within 48 hrs. This should be done in conjunction with
LD and AST assays to give a full picture as the test
has some drawbacks as the rise in plasma Ck Mb is
relatively transient. Also, CK Mb methods are
often less reliable and more expensive than other
enzyme tests.
 CK-MB
In the following circumstances, plasma Ck MB measurements
are strongly recommended.
When very early confirmation of the diagnosis of MI is
required ie. within 6 Hr.
When investigating post-operative patients for suspected MI.
In these patients, plasma CK MB remains normal in the
absence of myocardial damage, whereas AST and LD
activities are often increased in plasma for non-cardiac
diseases.
In patients suspected of having a second MI within a few
days of the first. It is easier to show that a second rise in
plasma enzyme activity has occurred if the activity of the
enzyme that is being measured rises rapidly after the
previous incident involving myocardial damage.
When it is suspected that an increased plasma total CK
activity may be due to release of the enzyme from skeletal
muscle (eg. after an intramuscular injection).
TIME COURSE OF PLASMA ENZYME
CHANGES AFTER MYOCARDIAL
INFARCTION
 Muscular Diseases
Because CK is the most active in skeletal muscle,
this enzyme is a sensitive indicator of disease.
CK isoenzyme changes in this group of diseases
are usually limited to CK-MM
New Cardiac Markers for
Myocardial Infarction
 New Cardiac Markers
Myoglobin: is the earliest new cardiac marker
available. It is a small molecule which passes rapidly
through damaged cell membranes. It can be detected
in serum within 2 to 5 hours of MI, peaks around 24 hr,
but falls to within its reference interval within 24 hr.
False positives can arise due to skeletal muscle
damage and are also seen in renal failure due to an
inability to excrete this molecule in the urine.

Troponin T and particularly Troponin I are specific

markers of myocardial damage. Their major
disadvantage is that serum abnormalities are not
detected unitl 6-18 hr after the onset of symptoms and
rise in parallel with CK-MB markers.
Alkaline Phosphatase isoenzymes
The alkaline phosphatases are a group of enzymes
that split off a terminal phosphate group from an
organic phosphate ester in alkaline solution. ALP
isoenzymes separated by acrylamide
electrophoresis have been identified in:
- liver
- intestinal mucosa
- placenta
- bile
- bone: it is in the diagnosis of bone diseases that
levels of ALP isoenzymes are of the greatest
clinical significance.
Separation by electrophoresis
 Separation by Physical Properties

Treatment Liver Bone Intestine

Heat Treatment- Relatively stable- *Inactivated Relatively stable

56oC, 15 mins (about 30-40%
remains)

L-Phenylalanine Relatively stable- Relatively stable- 95% inhibition

(25% inhibition) (25% inhibition)
 Clinical Significance
Increased Activity: serum ALP activity is raised in all bone
disorders accompanied by increased osteoblastic
activity. This includes Pagets disease, osteoblastic
tumours with metastases. It is also raised in primary and
secondary liver malignancies.

Decreased Activity- low levels of ALP enzymes are found in

a rare congenital defect, hypophostasemia in dwarfs
because of depressed osteoblastic activity, in
hypothyroidism and in pernicious anaemia.
Acid Phosphatase
 Acid Phosphatase Isoenzymes

ACP is concentrated in the prostate gland, at 100-300X the

concentration found in other tissues, such as leucocytes,
red blood cells and platelets. Up to 14 isoenzymes have
been described. Of these only two, the prostatic
isoenzyme is of clinical importance.
Separation and Quantitation:
1. Electrophoresis- on polyacrylamide gel is the method
of choice for classification of ACP isoenzymes. After
separation on an acidic polyacrylamide gel, the
phosphatases of the gel convert the diazo dye to form an
insoluble coloured product permanently deposited at the
location of each phosphatase enzyme.
Acid Phosphatase Isoenzymes.
 Acid Phosphatase Isoenzymes

Immunochemical Methods- RIAs and ELIZA

methods are the most specific methods for
detecting prostatic ACP in serum and
commercial kits are available for both of these
methods. Band 2 & band 4 are antigenically
similar. However, the quantity of ACP is 1000X
higher in the prostate than in other tissues,
 AMYLASE
These are hydrolases that split complex carbohydrates containing a-
D-glucose units linked through carbon atoms 1-4. They are able
to hydrolyse both linear and branched polyglucans.
Two types of amylases are recognised, but only one is of clinical
significance.
1. -amylase- eg. plant and bacterial exoamylase, acts only at the
terminal reducing end of a polyglucan chain; it splits of 2
maltose units at a time.
2. -amylase- or animal amylase, these are also called
endoamylases as they attack a-1-4-linkages in a random
manner, anywhere along a polyglucan chain. Large
polysaccharide molecules are thus rapidly broken down into
small units.
Amylose Maltose
(linear)
Amylopectin/ Glycogen limit dextran +
(branched) maltose

 Causes of High Amylase levels:

Pancreatic disorders- Acute pancreatitis, pancreatic
trauma or carcinoma.
Non-pancreatic abdominal disease-
a. Leakage of enzymes form the gut lumen to the
peritoneum: perforated ulcers, intestinal obstruction,
ischaemia of the small bowel.
b. Ruptured ectopic pregnancy.
Miscellaneous:
a. Salivary glands: mumps, duct obstruction
b. Carinoma of (bronchus, ovary, colon): amylase produced
by tumour.
c. Renal failure: decreased renal excretion.
d. Diabetic ketoacidosis.
e. Macroamylasaemia: amylase bound to a plasma protein
and unable to be excreted by the kidney.
f. Opiate drugs: morphine causes spasm of sphincter
 Measurement of Amylase Activity
Specimen- the serum enzyme is quite stable;
activity loss is negligible at room temperature in the
course of a week or at 4oC for up to 2 months. In
urine, an acid pH may make the enzyme less
stable, therefore the pH should be adjusted to 7
prior to storage.
Methods:
There are essentially 2 main methods:
1. Amyloclastic the rate of disappearance of the
substrate starch is measured. These methods were
popular in the 70's, but very few laboratories use
these methods today.
2. Saccharogenic- the rate of appearance of product
ie, free reducing groups; or, in the case of dyed
substrate methods, low molecular weight mono- and
oligo-saccharide fragments more soluble than the
polysaccharide substrate.
 CHOLINESTERASE

There are also 2 cholinesterases, the true

acetylcholinesterase found in nervous tissue and
the pseudo one found in liver, heart, muscle
and plasma, which catalyses the hydrolysis of
choline esters. This pseudocholinesterase is the
most useful clinically.
 Causes of Decreased Plasma
Pseudocholinesterase
1. Inherited abnormal variants (suxamethonium sensitivity).
2. Secondary Causes-
I. Liver disease: decreased synthesis
ii.Organophosphate toxicity (insecticide poisoning)
iii. Severe anaemia
iv. Myocardial infarction
Suxamethonium sensitivity: the muscle relaxant suxamethonium is
destroyed by pseudocholinesterase. In subjects with cogenitally
decreased levels and abnormal variants of this enzyme prolonged
apnoea follows its administration in anaesthesia..

 THE AMINOTRANSFERASES
(TRANSAMINASES)

The aminotranferases constitute a group of

enzymes that catalyse the interconversion of
amino acids and -oxo-acids by transfer of
amino groups. The two enzymes with the
greatest clinical importance are Aspartate
Transaminase (AST) and Alanine
Transaminase.
 Aspartate Transaminaase
AST
2-oxoglutarate + L-aspartate L-glutamate + oxaloacetate

The oxaloacetate produced is simultaneously reduced by NADH and

malate dehydrogenase to malate:
Malate dehydrogenase
Oxaloacetate + NADH + H+ Malate + NAD+

The decrease in the NADH concentration is directly proportional to the

AST activity of the sample and is determined by measurement of the
rate change of absorbance at 340 nm. This is the standard or
reference method and is used whether in a =n automated or manual
fashion in most clinical labs.
 Myocardial Infarction
Increased AST activity appears in serum. On
average levels do not become abnormal, until 6-
8 hours has elapsed after the onset of chest
pain. Abnormal AST levels are seen in 97% of
cases of myocardial infarction when correctly
timed blood specimens are analysed. Peak
values are reached after 18-24 hours, and the
activity falls to within normal range by the 4th or
5th day. The peak values of AST are roughly
proportional to cardiac damage. Levels of 10-15
times normal are often associated with fatal
attacks.
 Liver Diseases
In viral hepatitis and other forms of liver disease associated with
hepatic necrosis, serum AST and alanine transferase are
elevated by roughly equal amounts even before clinical signs of
the disease such as jaundice appear. Levels for both enzymes
may reach 100 times the upper reference limit, although 20- to
50- times are more often encountered. Peak values of AST occur
by the 3rd to 5th week if no complications occur.

The relatively similar elevations of AST and ALT in hepatitis have

been attributed to the release of only the cytoplasmic isoenzyme
of AST into the circulation from reversibly damaged parenchymal
cells of the liver. However, when necrosis of cells occurs,
considerably higher amounts of mitochondrial AST are also
released and higher amounts of AST relative to ALT are
recorded. This AST/ALT ratio is therefore useful in assessing the
severity of the disease.
 Muscular Diseases
AST activity levels are increased in progressive
muscular dystrophy reaching up to 8 times
normal. They are however usually normal in
other muscular diseases.
 ALANINE AMINOTRANSFERASE
There are no isoenzyme forms of ALT.
ALT
2-oxoglutarate + L-alanine L-glutamate +Pyruvate

The pyruvate is simultaneously reduced by NADH and lactate

dehydrogenase (LD) to lactate:
LD
Pyruvate + NADH + H+ L-lactate + NAD+
The decrease in the NADH concentration is directly proportional to the
ALT activity of the sample and is determined by measurement of the
rate of change of absorbance at 340 nm. Once again this is the
method of choice for measuring the activity of ALT in serum samples
in the clinical laboratory.
 Clinical Significance

De Ritis ratio: (ALT/AST): The concentration of

ALT in tissues is not nearly as great as for AST.
It is present in moderately high concentrations in
the liver, but is low in cardiac and skeletal
muscles and in other tissues. Its use for clinical
purposes is primarily for the diagnosis of liver
diseases, and ALT is considered the more liver-
specific enzyme. Serum elevations of ALT are
rarely seen in any disease other than
parenchymal liver disease. Moreover, elevations
of ALT activity persist longer and are usually
higher than those of AST activity.

Acute Viral Hepatitis

 TESTS OF LIVER CELL INTEGRITY- (-
glutamyltranspeptidase [GGT] )
This enzyme occurs in the plasma membrane of many
cells, and transfers a -glutamyl residue from one
peptide to another.
Its normal functional role is unclear, but it has been
suggested that it might :-
assist the transport of peptides into cells, or
play a role in glutathione metabolism
(glutathione = -glutamyl cysteinyl glycine)..
It is measured using a synthetic substrate :-
-glutamyl-p -nitroanilide + glycyl glycine (acceptor)
GGT, pH 8.2
-------------> g-glutamyl glycyl glycine + p -nitroaniline
 TESTS OF LIVER CELL INTEGRITY- (-
glutamyltranspeptidase [GGT] )
This substrate (-glutamyl-p -nitroanilide) is only
sparingly soluble in water, and optimal concentrations
cannot be achieved. In recent years, a more water-
soluble substrate has come into use, and allows higher
activities to be obtained. It is -glutamyl-3 carboxy-4-
nitroanilide.
GGT is easily induced by alcohol, and various drugs.
Thus, the Reference Interval for males is > for females,
possibly due to greater alcohol consumption in the
former.
It is very sensitive to obstructive liver disease (more
than ALP) and highest values are seen in intra- or post-
hepatic biliary obstruction.
GGT is only mildly elevated in infectious hepatitis
(hepatitis A) (less useful here than the
aminotransferases).