Biochemistry. Enzymes.

Nino Gulatava
December,2020
Enzymes
Enzymes are biological materials with catalytic properties, i.e. they
increase the rate of chemical reactions in cells and in vitro systems
that otherwise proceed very slowly.
Enzymes are large, naturally occurring proteins with molecular
weights usually between 13000 and 500000.
The study of these molecules has became a valuable diagnostic tools
for the elucidation of various disease states for testing organ function.
Many enzymes were first named for their function (e.g. Lactate
dehydrogenase), but some were named for the type of substrate on
which they act: urease hydrolyzes urea, lipase hydrolyzes lipids,
phosphatases act on organic phosphates.
Enzymes
All enzymes are divided into one of six general classes depending on type of reaction
they catalyze.
• Oxidoreductases: Lactate dehydrogenase(LDH),Malate dehydrogenase (MDH),
Ferroxidase (ceruloplasmin).These enzymes catalyze electron transfer or oxidation-
reduction reactions.
Ared + Box=Aox+ Bred
• Transferases : -Glutamyl transferase (GGT), Aspartate aminotransferase
(AST/SGOT), Alanine aminotransferase (ALT/SGPT), Hexokinase, Pyruvate kinase,
Creatin kinase (CKL). These group enzymes catalyze the transfer of a group (e.g. an
amino, carboxyl ..) from one molecule to another.
A-X+B=A+B-X
• Hydrolases: Cholinesterase, Lipase, Alkaline phosphatase (ALP), Acid phosphatase,
alpha-Amylase (Diastase), Chymotrypsin. These enzymes catalyze the cleavage of
C-O, C-N, C-C and some other bonds with the addition of water.
A-B +H2O A-OH + B-H
Enzymes
• Lyase: Fructose-biphosphate aldolase ( Aldolase), Porphobilinogen aldolase.These
enzymes catalyse C-C,C-O and C-N bonds by elimination, with the formation of a
double bond or the reverse reaction , the addition of a group to a double bond . In case
where the reverse reaction is important the term synthase is used in the name.
A+BAB or AB  A+B
• Isomerases : Glucosephosphate isomerase, Triosephosphate isomerase. These group of
enzymes catalyse structural or geometric changes in amolecula. Thee may be called
epimerase, isomerase, mutases depending on the type of isomerism involved.
ABC  CAB
• Ligases: Glutamine synthetase – in this case two molecules are joined , coupled with
hydrolysis of the pyrophosphate in ATP. These group none use in clinical diagnostic.

A+B+ATP  AB+ADP+Pi
Enzymes
All enzymes are proteins; that is, they are complex compounds of
high molecular weight. The y contain amount of carbon, hydrogen,
oxygen, nitrogen, and sulfur that are similar to amounts found in
other proteins materials. Enzymes are distinguished from other
proteins by their catalytic action.
The catalytic behavior of an enzyme depends on the primary,
secondary, tertiary, and quaternary structures of the protein
molecule. Changes to the primary amino acid sequence usually result
in the differences in the three dimensional structure because the
secondary and tertiary folding end up different. However, changes to
any one of these structures can effect the enzymatic activity of the
protein, usually reducing or abolishing it.
Measurement of enzymes
The reaction rates of most enzymatic procedures are not constant. But
observing the rate of change of absorbance for a starting material or
product at a specific wavelength, the reaction can be followed. When the
reactants are mixed and reach thermal and kinetic equilibrium, a lag phase
occurs with a little change of absorbance per unit time. Then a linear
phase of constant absorbance change per unit time occurs, and finally a
substrate depletion phase with a little change of absorbance per unit time.
Enzyme assays must be performed during the linear phase of absorbance
change, where a constant amount of activity can be determined for a
period of time. Measurements do not start at zero time but after the lag
phase has occurred. Measurements can be made at any time during the
linear phase and can continue up to the substrate depletion phase.
Measurement of enzymes
Enzymes are measured by the
two most common techniques –
end-point at a fixed time and
kinetic method at multi-point
fixed time.
End-point method – measure the
amount of an analyte after no
further reaction is occurring.
If the rate of reaction is followed
continuously or with many
points as a function of time, the
assay is termed a kinetic assay.
Measurement of
enzymes
Analytical factors affecting
measurement –pH, temperature,
concentration of substrate, buffer,
cofactors, activators, inhibitors,
coupling enzymes.
AST/SGOT
Normal value : 10-38 U/L.
It is widely distributed in tissues - the heart, the liver, the musculature musculature.
AST has mitochondrial and cytoplasmic isoenzymes. In serum in normal condition
we have cytoplasmic isoenzyme AST.
•  AST/SGOT:
• Heart stroke (Myocardial infarction ) - 93-98 %.In this case increase of level of
AST is start after 6-8 h and max of concentration after 24-36 h and return to
normal value on 5-6 days.
• Hepatatis – acute, but more symptomatic for deep damage of hepatocytes
• Coefficient of Rittes - AST/ALT=1.33
• If > 1.33 – we have disease of heart
• If < 1.33 – we have problem with liver
ALT/SGPT
Normal value : 7- 41 U/L
ALT also distributed in scelet muscle, liver, heart, but most of all concentration in liver.
 ALT/SGPT:
Hepatitis – acute hepatitis. This enzymes more sensitive and than early diagnostic test for acute
hepatitis than AST. ALT is mainly located in cytoplasm of cells, AST - in mitochondria.
According this fact, ALT is marker of acute damage, AST – for severe damage of hepatocyte.
Increase of level both enzymes are 15 days before jaundice in viral hepatitis A, and 3-4 weeks
before in viral hepatitis B. The level of ALT is more than AST and coefficient of Rittes=0.55 –
0.65 . If we have severe damage of liver coefficient is more 0.83.
IF we have alcoholic damage of liver - coefficient Rittes is more than 2.0
ALT, AST:
• 1.5-5.0 times - moderate increase
• 6- 10 times – medium damage
• 10 times - high damage .
Lactate Dehydrogenase (LDH)
Normal value: 240 – 840 U/L.
Most of activity of LDH was discovered in kidney, heart muscle, skeletal muscle, liver. LDH is contains
not only in serum, but in erythrocytes also in significant amount, that why is very important a sample
for investigation should without any hemolysis. Most of organs and tissue has five isoenzymes of LDH.
Heart, brain, kidney – LDH-14, LDH-2; Liver, skeletal muscle – LDH-4; LDH-5. In human serum all of
isoenzymes are present , but there are pattern of activity these isoenzymes: LDH-2> LDH-1>LDH-3>
LDH-4> LDH-5.
In healthy persons may be increase LDH activity in some conditions:
Pregnancy, newborn, after intensive physical activity.
•  LDH:
• Heart stroke – 2-4 times more after 8-10 h of stroke. Max concentration of LDH after 48-72 h and still
stay increased during 10 days.
• Lung stroke and emboli
• Myopathy- muscle dystrophia, traumatic damage of muscle, inflammatory process, metabolic and
endocrine disease
• Acute viral hepatitis- at the first days of jaundice
• Anemia – hemolytic, megaloblastic. This enzymes use for differential diagnosis of Jilber disease (LDH
is norm) and chronic hemolytic anemia( LDH is ).
• Acute and reactivation of chronic diseases of kidney.
Gamma-Glutamiltransferase (GGT)

Normal value: female 5 – 36 U/L, male - 8 – 61 U/L.

GGT was discovered in liver, pancreas, kidney.
This is specific enzyme for liver- more than ALT, AST,ALP.
Especially more sensitive for alcoholic damage of liver. Activity of
GGT is hepatotoxic marker and positive in 90% of cases of liver
disease.
 
Alkaline Phosphatase (ALP)

Normal value: adult (31 y) - 39-92 U/L; adult ( > 31 y) – 39 – 117 U/L.
ALP is located on intestinal mucosa, osteoblasts, on the walls of bile ducts in liver.
 ALP:
• Destruction and damage of liver – viral and autoimmune hepatitis, toxic and medicaments damage of liver
• disorders of transport bile - extra hepatic obstruction of bile ducts - stone , post surgery stricture, the
primary sclerotic cholangitis
• Cirrhosis of liver
• Cholestasis
• Cancer of liver
• Treatment by some of medicaments: tetracycline, paracetamol, mercaptorune, salicylates and etc.)
• Per oral contraceptive
• Bone disease - Healing of fractures, osteogenic carcinoma, metastasic damage of bone, myeloma disease,
lymphogranulomatosis with bone damage
• osteomalation
• hyperparathyroidism
• disease of intestine
• sepsis
• thyrotoxicosis
• rachitic disease in children
Alkaline Phosphatase (ALP)

¯ALP:

• hypothyreosis
• anaemia
• hypophospathemia
Cholinesterase

Normal value: 5320-12920 U/L.

In human tissues, two different enzymes of this type have been
found: 1, acetylcholinesterase (true cholinesterase), which is
predominantly in the nervous tissue, skeletal muscles and in low
concentration in erythrocytes and 2, serum or pseudocholinesterase,
which is widespread, present in the liver, pancreatic Gland, secrets
biscuits into the blood. Serum catalyzes the hydrolysis reaction of
acetylcholine.
Determination of the activity of this enzyme is of clinical interest for
the diagnosis of poisoning with phosphorogenic substances and
insecticides, and also as an indicator of the protein-synthesizing
function of the liver.
Cholinesterase

Cholinesterase:
• chronic severe liver disease
• With viral hepatitis in the development of acute hepatic insufficiency
• At a myocardial infarction by the end of the first day of the disease

•  Cholinesterase:
• nephritic syndrome
• obesity
• arterial hypertension
• diabetes mellitus, chorea
• Manic-depressive psychosis
• Depressive neurosis
• anxiety
Alpha-Amylase

Normal value: 28 - 100 U/L.

Alpha-Amylase is catalyze hydrolysis of polysaccharides to simple mono- and
disaccharides. Most of all alpha amylase are in pancreas and salivary glands.
Plasma of human blood contains two types - pancreatic and salivary. In physiological
conditions, amylase of blood serum contains 40% of the pancreatic and 60% of the
salivary gland.
Detection of activity this enzyme is very important for diagnostic of pancreas disease.
The increase in activity is twice estimated as a symptom of a pancreatic lesion.
With urine, mainly P-amylase is released, which is the reason for the high information
about the functional state of the pancreas uroamylase than the amylase of the blood
serum. It is believed that 65% of the urine amylase activity is due to pancreatic amylase.
In acute pancreatitis, its content increases in serum to 89% And urine to 92% without
changes in salivary gland parameters. Hyperamylasemia occurs at the beginning of the
disease (after 4-6 hours), reaches a maximum after 12-24 hours, and then quickly
decreases and comes back to normal on the 2-6 day.
Acute pancreatitis can occur without an increase in amylase - with pancreonecrosis. It is
important to study P-amylase in the daily urine and repeat the test within two days.
Lipase

Normal value: 13 - 60 U/L.

Lipase is enzyme which is catalyze glycerides to glycerin fat acids.
Most important lipase of pancreas. This enzymes is used for
diagnostic of acute pancreatitis. The content of lipase increases as the
alpha amylase and persists for a long time than alpha amylase
In acute pancreatitis the activity of lipase increases during several
hours, reaching a maximum of 12-24 hours and remains elevated for
10-12 days.
In contrast to amylase lipase does not increase with parotitis, ectopic
pregnancy, lung cancer, peritonitis, biliary colic, bone fractures, soft
tissue injury, after surgery, in breast cancer.