ENZYMES
Enzymes - proteins - speed up biochemical reactions
1 molecule is transformed noncatalyzed in comparison to106-
1014 molecules transformed via catalysis by enzyme
The mechanism of action of enzymes consists of reducing the
activation energy for the reaction (enable course of
metabolic processes at relatively low temperatures (37 ° C)
and at pH 6.5 to 7.5 in an aqueous medium)
There are from 1000 to 4000 different types of enzymes
in animal cell
protein part - apoenzyme, nonproteinic – coenzyme
reactant - substrates, the substances formed –products
Reaction: The substrate binds to the specific binding site on
the enzyme molecule (binding site is wedged into the recess
enzyme called. Active center)
Pathobiochemistry- enzymes and inhibitors 1
The enzymatic reaction-principle
Binding of substrate - enzyme:
- by non-covalent ionic bond
- hydrogen and hydrophobic bridges
- van der Waals interactions
• On the functional groups of aminoacids residues in
active site of enzyme are linked coenzymes, eventually
metal atoms (metaloenzymes), participate in the catalyzed
reaction
• Functional groups activate the substrate and reduce the
energy which is required to produce high-energy
intermediate stage
Pathobiochemistry- enzymes and inhibitors 2
Binding of the enzyme-substrate
http://www.google.cz/url?sa=i&rct=j&q=&esrc=s&source=imag
es&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http%3A%2F
%2Ffaculty.samford.edu%2F~djohnso2%2F44962w%2F405%2
Fmetabolism.html&ei=meLyVPKHJcf5UqDMgMgP&bvm=bv.
87269000,d.d24&psig=AFQjCNFd4SZ50y4rttbYAjGKliZh-
KU7fA&ust=1425290217916178
Pathobiochemistry- enzymes and inhibitors 3
Pathobiochemistry of regulation enzyme activity
Inhibition of enzyme activity
The effect of multiple drugs or toxins resides in its
ability to inhibit the enzyme.
Strongest inhibitors - bind covalently to functional
groups of the active site, eventually substrate
analogs - forming enzyme complexes
The rate of enzyme reaction :
a) Concentration of substrate, product
b) Concentration of activators, concentration od
inhibitors
The relationship between the rate of the enzyme
reaction and the substrate concentration - given by
Michaelis-Menten equation
E + S ← → ES → P + E
Pathobiochemistry- enzymes and inhibitors 4
Products and physiological inhibitors may compete with the
substrate for binding to the active center of the enzyme
and thus slow the rate of reaction
Physiological regulation of metabolic pathways– the ability to
alter the rate of progression of metabolic reactions in the
pathway via activation of enzymes that catalyze the slowest
part
Enzymes have so called allosteric activators or inhibitors i.e.
compounds which bind to a different part of the enzyme
molecule than the active site thus affecting the
conformation of the enzyme molecule
- multimeric enzymes, CAC enzymes
The regulation also – by modulatory protein or phosphorylation
Isoenzymes: enzymes having a different amino acid sequence
in the peptide chain, but catalyzing the same reaction
Pathobiochemistry- enzymes and inhibitors 5
INHIBITORS:
The activity of many enzymes can be inhibited by the binding
of specific small molecules and ions. This means of inhibiting
enzyme activity serves as a major control mechanism in
biological systems.
The regulation of allosteric enzymes typifies this type of
control.
In addition, many drugs and toxic agents act by inhibiting
enzymes. Inhibition by particular chemicals can be a source
of insight into the mechanism of enzyme action: specific
inhibitors can often be used to identify residues critical for
catalysis.
Enzyme inhibition can be either reversible or irreversible.
An irreversible inhibitor dissociates very slowly from its
target enzyme because it has become tightly bound to the
enzyme, either covalently or noncovalently.
Some irreversible inhibitors are important drugs.
Pathobiochemistry- enzymes and inhibitors 6
Mechanisms of regulation of the enzyme activity -
INHIBITION
Irreversible inhibition
Reversible inhibition of the active center
The enzyme inhibitor is a compound that reduces the
rate of response by binding to the enzyme. Reversible
inhibitors – there is not a covalent bond, can be
detached from the enzyme. Products - are reversible
inhibitors for their own reaction.
Competitive inhibition
Reversible inhibitor may compete for binding to the
active center with the substrate, producing an enzyme
complex that may be dissociated into free enzyme and
inhibitor.
Non-competitive inhibition
This inhibitor does not compete for the binding site, but
its binding to the enzyme reduces the concentration of
active enzyme, i.e. always decreasing Vmax
Pathobiochemistry- enzymes and inhibitors 7
Scheme of inhibition
In competitive inhibition of the binding site for the substrate A
competes with structurally very similar other substrate.
In non-competitive inhibition substrate A gets to its binging site, but at
the position on the binding site of its partner i.e. substrate B occupies a
non-competitive inhibitor (to substrate A), but that is competitive with to
B.
In non-competitive inhibition the inhibitor binds to the enzyme-substrate
complex.
http://images.slideplayer.co
m/1/277615/slides/slide_53
.jpg
Pathobiochemistry- enzymes and inhibitors 8
Examplex of inhibition
Non-competitive inhibition
•Non-competitive inhibitor binds only to the
enzyme-substrate complex (when the enzyme binds
substrates cotrollably):
•the first substrate molecule induces a change in the
conformation of the enzyme molecule, the binding
site for either the co-substrate or inhibitor openes.
Non-competitive inhibitor reduces Km and Vmax, as
well.
Competitive inhibition
Molecules of inhibitor - structurally similar to the
substrate that covalently bind so tightly in the
active center that this binding can not be displaced.
This way - a common mechanism of action of drugs
or antimetabolites
Pathobiochemistry- enzymes and inhibitors 9
Pathobiochemistry- enzymes and inhibitors 10
The competitive inhibitor
resembles the substrate and binds to the
active site of the enzyme (Figure 8.15). The
substrate is thereby prevented from
binding to the same active site. A
competitive inhibitor diminishes the rate of
catalysis by reducing the proportion of
enzyme molecules bound to a substrate. At
any given inhibitor concentration,
competitive inhibition can be relieved by
increasing the substrate concentration.
Under these conditions, the substrate
“outcompetes” the inhibitor for the active
site. Methotrexate is a structural analog of
tetrahydrofolate, a coenzyme for the
enzyme dihydrofolate reductase, which
plays a role in the biosynthesis of purines
and pyrimidines (Figure 8.16). It binds to
dihydrofolate reductase 1000-fold more
tightly than the natural substrate and
inhibits nucleotide base synthesis. It is used
to treat cancer.
Examples of irreversible competitive inhibition
Inhibitor Enzyme Effect
Aspirine cyclooxygenase Anti inflammatory
Allopurinol xanthinoxidase Treatment of gout
5-Fluorouracil thymidilatesynthase cancerostatic
Penicilin transpeptidase antibiotics
Sarin cholinesterase Nerve gas
ß-aminopropionitrile lysyloxidase lathyrism
Pathobiochemistry- enzymes and inhibitors 11
Mechanism of action of irreversible inhibitors
Affinity changes of binding site – the substrate
analog has a reactive group that is not the natural
substrate and which permanently blocks the active site
for substrate (covalent bond with an amino acid
residue)
!!! resistance to antibiotics
Excessive and prolonged use of antibiotics also in
agriculture, veterinary practice… have made some
bacterial strains - Pseudomonas,
Streptococcus,Staphylococcus, Mycobacterium
tuberculosis… resistant to some antibiotics
The mechanism of this resistance may be due to
induction of the synthesis of enzymes, which modify
the antibiotic molecule so that it becomes analog of
the transitional stage.
Pathobiochemistry- enzymes and inhibitors 12
Example of resistance
Induction of ß-lactamases are altered so called ßlactam
antibiotics :
penicillins, cephalosporins, carbapenems
A similar effect may have the induction of:
acetyltransferase, fosfotransferase or
nukleotidyltransferase on aminoglykosides
acetyltransferase on chloramphenicol
Pathobiochemistry- enzymes and inhibitors 13
Allosteric regulation
Allosteric effector reversibily binds to another than binding site of
the enzyme. This bond induces: conformational change of the active
site, so it may activate or
inhibit the enzyme
Allosteric regulation
regulation mechanisms of enzyme activity
Scheme of
allosteric
activation
and
inhibition
http://academic.
pgcc.edu/~krobe
rts/Lecture/Chap
ter%205/05-
11_AllostericCo
ntrol_L.jpgPathobiochemistry- enzymes and inhibitors 14
Other regulation mechanisms of enzyme activity
Covalent modifications – phosphorylation of hydroxyl group by
phosphorylasakinase. Phosphorylation of enzyme activates the
active site of enzyme → activ conformatin, dephosphorylatin →
inaktivation.
Limited proteolysis - aktivation induced by cleaving the short
polypeptide chain from the peptide, e.i. proenzyme or enzymogene
leads to change in the conformation of the active site that can
bing the substrate in this form.
Examples:chymotrypsinogen ® chymotrypsin
prothrombin ® thrombin
Note: - proteolytic pancreatic enzymes, which are formed in the
pancreas as prodrugs, are activated in the gut lumen.
Otherwise, they would digeste own pancreatic tissue.http://www.worthington-
biochem.com/chy/images/reaction.jp
g
Pathobiochemistry- enzymes and inhibitors 15
Other regulation mechanisms of enzyme activity
Induction of enzyme synthesis
the amount of enzyme in the cell depends on the rate
of its synthesis or degradation (lasts several hours or
days, previous methods of control activities last only)
Suppression synthesis of the enzyme
Example: by this mechanism acts e.g. omeprazole – suppresses
protein synthesis e.i. proton pump in the gastric mucosa, thus
preventing the production of HCl. It is used to reduce the
acidity of gastric secretion, thereby limiting its aggressive
action (treatment of peptic ulcer disease).
feedback regulation
final product regulates the speed of its own synthesis,
the final product of a metabolic pathway may inhibit or
related metabolites may activate regulatory key)
enzyme
final product may regulate the speed of its own synthesis - action
on gene transcription key enzyme in the metabolic pathway – much
more slower process than regulation by allosteric mechanism.
Pathobiochemistry- enzymes and inhibitors 16
Classification and nomenclature of enzymes
Classes of enzymes:
The ending of enzyme names: substrate + -ase
e.g. lactate dehydrogenase, amylase, alcohol
dehydrogenase, aspartate transaminase…
Pathobiochemistry- enzymes and inhibitors 17
•1 Oxidoreductases
•2 Transferases
•3 Hydrolases
•4 Lyases
•5 Isomerases
•6 Ligases
•7 Translocases
Pathobiochemistry- enzymes and inhibitors 18
Oxidoreductases catalyze oxidation-reduction reactions where electrons are transferred. These electrons are usually in
the form of hydride ions or hydrogen atoms. When a substrate is being oxidized it is the hydrogen donor. The most
common name used is a dehydrogenase and sometimes reductase will be used. An oxidase is referred to when the oxygen
atom is the acceptor.
Transferases catalyze group transfer reactions. The transfer occurs from one molecule that will be the donor to another
molecule that will be the acceptor. Most of the time, the donor is a cofactor that is charged with the group about to be
transferred. Example: Hexokinase used in glycolysis.
Hydrolases catalyze reactions that involve hydrolysis. This cases usually involves the transfer of functional groups to
water. When the hydrolase acts on amide, glycosyl, peptide, ester, or other bonds, they not only catalyze the hydrolytic
removal of a group from the substrate but also a transfer of the group to an acceptor compound. These enzymes could
also be classified under transferases since hydrolysis can be viewed as a transfer of a functional group to water as an
acceptor. However, as the acceptor's reaction with water was discovered very early, it's considered the main function of
the enzyme which allows it to fall under this classification. For example: Chymotrypsin.
Lyases catalyze reactions where functional groups are added to break double bonds in molecules or the reverse where
double bonds are formed by the removal of functional groups. For example: Fructose bisphosphate aldolase used in
converting fructose 1,6-bisphospate to G3P and DHAP by cutting C-C bond.
Isomerases catalyze reactions that transfer functional groups within a molecule so that isomeric forms are produced.
These enzymes allow for structural or geometric changes within a compound. Sometime the interconversion is carried out
by an intramolecular oxidoreduction. In this case, one molecule is both the hydrogen acceptor and donor, so there's no
oxidized product. The lack of a oxidized product is the reason this enzyme falls under this classification. The subclasses
are created under this category by the type of isomerism. For example: phosphoglucose isomerase for converting
glucose 6-phosphate to fructose 6-phosphate. Moving chemical group inside same substrate.
Ligases are used in catalysis where two substrates are ligated and the formation of carbon-carbon, carbon-sulfide,
carbon-nitrogen, and carbon-oxygen bonds due to condensation reactions. These reactions are couple to the cleavage of
ATP.
Translocase are enzymes that catalyze the movement of ions or molecules across membranes or their separation within
membranes. It is a general term for a protein that assists in moving another molecule, usually across a cell membrane. The
reaction is designated as a transfer from “side 1” to “side 2” because the designations “in” and “out”, which had previously
been used, can be ambiguous. Translocases are the most common secretion system in Gram positive bacteria.
The causes of increased activity, enzyme concentration in
plasma
1. Patological ↑ conc. of enzymes in plasma – the result of the
increased permeability of the cell membrane - damaged by chemicals,
anoxia, hypoxia, viruses, inflammation. May lead to cell degradation.
Cell degradation→ Increased phospholipase activity, the degradation of
cytoplasmic membrane phospholipids → perforation→ release of
contents + enzymes into extracellular space → plasma
2. Incerased synthesis E – is not pathological, but is associated with a
condition in organism :
e.g. bone growth ↑ osteoblasts activity↑ alkaline phosphatase in
blood
In children ALP 3x-7x higher than in adults
3. Drugs, alcohol ↑ enzyme activity (liver) – ALP, GMT etc.
Pathobiochemistry- enzymes and inhibitors 19
The causes of increased enzyme concentration in
plasma
3. Release from cells not associated with cell death or increased
synthesis –
E.g. ethanol releases expression of mitochondrial AST in hepatocytes,
its transport to the surface hepatocytes → release into blood.
E.g. Food intake→ intestinal ALP →into lymph, ↑ in blood.
E.g. Liver enzymes are bound to the surface of hepatocytes ….
4. Some cases of increased concentration – inadequate removal from
circulation.
E.g. Small enzymes– amylase, lipase – removed from circulation by
glomerular filtration.
Renal impairment, renal failure ↑ their concentration in the blood.
Formation of complexes E-Ab, macroenzyme, half-life Ig-3 weaks
Pathobiochemistry- enzymes and inhibitors 20
Time course of the increase and decrease of enzyme activity in plasma
Time course – influenced by many factors:
During apoptosis the cell membrane defects deepen
with time. Result – from cells are first released small
molecules of enzymes and later large enzymes.
E.g. myocardial infarction (IM) – at first in plasma AST
and CK (small molecules), later LD (bigger). In IM –
concentration of CK in plasma depends on the size of the
bearing affected by IM.
When the cause of cell damage disappear enzyme
concentration persists for some time, then decreases.
E.g. acute hepatitis can be distinguished from toxic liver
damage. At virus hepatitis – imunological cell damage,
longer persistance of increased enzyme concentration,
toxic damage – faster return to normal levels of the
enzyme (GGT,AST, ALT).
Pathobiochemistry- enzymes and inhibitors 21
The activity of enzymes in plasma
Enzyme concentration gradient between the cell and
plasma– inside hepatocytes in cytoplasm = higher
concentration of AST than ALT , LD minimum there.
• E.g. Hepatocyte damage- fastest-growing AST, but LD at
least.
• E.g. Myocardium – hogh concentration of CK, low of LD,
damage → hogh increase of CK in plasma
Enzyme concentration in plasma is determined by the rate
of its removal.
low molecular weight -glomerul. filtration
others (the majority) - inactivated in plasma, removed
by cells of the RES via receptor, endocytosis
Pathobiochemistry- enzymes and inhibitors 22
Definition - the half-life of enzyme
The period during which the enzyme is increased in plasma is
determined by its own half-life.
The biological half-life of enzyme – the period during which the
amount of enzyme decreases half, unless added from tissues.
Enzyme Half-life
ALP 3-7 days
AMS 9-18 hours
ALT 2 days
AST 12-14 hours
CHS 10 days
CK 10 hours
GMT 3-4 days
LDH1(HHHH) 4-5 days
LDH5(MMMM) 10 hours
Pathobiochemistry- enzymes and inhibitors 23
The use of enzymes in clinical diagnosis
Detection of tissue damage
Identification of the beginning of tissue damage
Determining the extent of damage
Estimation of the severity of cell damage
Diagnosis of basic disease
Specification diagnosis within the damaged organ
Pathobiochemistry- enzymes and inhibitors 24
• The main diagnostic hepatocellular enzymes are localized in different
areas of hepatocyte. ALT and cytoplazmatic isoenzyme AST are placed in
cytoplasm.
• When is the membrane damaged (e.g. viral or chemical), these enzymes are
released and entered the sinusoid. The result is an increase in plasma.
• Mitochondrial AST is primarily released during mitochondrial damage, egg.
when exposed to alcohol.
• ALP and GGT are located on the canalicular surface of hepatocytes and
released in cholestasis, particularly due to the effect of bile acids on the
membrane.
• GGT is also located in the microsomes, where is induced by certain drugs.
http://o.quizlet.com/i/eHnZCzz2IZU1GLwWW5FTw
g_m.jpg
Pathobiochemistry- enzymes and inhibitors 25
Specification of diagnosis
• Information suitable for precise diagnosis is obtained :
• - from values of the catalytic enzyme concentration in a body fluid (a direct correlation
between the degree of organ damage and increased activity of enzymes in blood).
• - spectrum of enzymes present in the blood (e.g. in severe liver damage accompanied by
cell necrosis is increased activity of enzymes in the blood subsequently : LD> AST> ALT).
• - calculating the ratio of the enzyme activities (e.g. according to the ratio of AST / ALT in
serum can be distinguished initial obstructive jaundice (AST / ALT <1) from chronic active
hepatitis (AST / ALT> 1). For acute disease can be from the ratio of enzyme activities
with short and long biological half-life to determine the stage of disease or predict the
course of the disease, e.g. in acute hepatitis decrease in
the ratio of AST (half-life 12 h) / ALT (half-life of 2 days) helps to qualify the type of
hepatitis.
• - monitoring the enzyme activity (mechanism of enzyme release into the blood from
damaged tissues and their clearance is characteristic curves of typical kinetic activity of
which can be derived during the period of time during which the disease may be present or
to determine the stage of disease)
• - determination of isoenzymes.
Pathobiochemistry- enzymes and inhibitors 26
Macroenzymes
• Macroenzymes are complexes formed by enzyme linked
immunoglobulin, a lipoprotein, protein, or cell membrane fragments.
• Generally macroenzymes have higher molecular weight and longer
half-life in blood. Macroenzyme´s presence in serum may affect
its analytical determination or cause misinterpretations of results.
• Macroamylasemia is a well-known example when the amylase
forms a complex with other macromolecules and therefore is not
filterable into urine. As a result, accumulates in the blood. The
level of amylase in the blood is then increased without causing its
increased release from damaged tissue (about 1-3% of patients
with elevated serum amylase).
• Because macroamylase does not penetrate into the urine, urine
amylase level is normal or reduced in this case. Unlike the situation
during e.g. pancreatitis when elevated levels of serum amylase is
accompanied by an increase of amylase in urine.
Pathobiochemistry- enzymes and inhibitors 27
Clinically important enzymes
Aminotransferases
provide the conversion of amino acids and keto
acids α-amino transfer, the donor and acceptor
amino group is a 2-oxoglutarate / L-glutamate
Alanine aminotransferase, ALT – donor -NH2 Ala to
form pyruvate, marker- liver (viral hepatitis,
alcohol, hepatopathy, ...)
• Aspartate aminotransferase, AST – donor – NH2 Asp
to form oxaloacetate, a marker of damage liver,
heart, myocardial infarction, muscular
damage
Pathobiochemistry- enzymes and inhibitors 28
Clinically important enzymes
-amylase, AMS
synthesized in the pancreas, cleaves α-1,4glycosidic
linkage in starch and glycogen to produce
maltose and maltotriose, endoglycosidase, marker acute
pancreatitis
alkaline phosphatase, ALP
hydrolysis of monoesters of phosphoric acid with
alcohols, phenols, Glycine - nonspecific - cleaves
POC, POP, PS, PN, has several isoforms according
to the synthesis of tissue - bone, liver, placenta,
intestinal, optimum in the alkaline environment,
marker - bone damage, liver
acid phosphatase, ACP
• properties ALP, optimum in the acidic region, marker-
prostate
Pathobiochemistry- enzymes and inhibitors 29
Other clinically important enzymes
creatine kinase , CK
catalyzes the reversible phosphorylation of creatine to
phosphocreatine for ATP consumption, a marker of muscle
damage, heart
lactate dehydrogenase, LDH
isoforms (tissue) of the heart, liver, catalyzes the reaction:
Lactate + NAD + ↔ pyruvate + NADH + H + (reversible) is nonspecific
tissue damage according isoforms - electrophoretically
(LDH3 pulmonary embolism, myocardial infarction LDH1,2,
hepatopathy + disease koster.svalstva LD4,5).
Pathobiochemistry- enzymes and inhibitors 30
Clinically significant enzymes
• Causes of increased activity in serum
• AST aspartate aminotransferase myocardial infarction; hepatopathia; blood diseases; muscle damage
• ALT alanine aminotransferase hepatic dysfunction, heart disease, AST / ALT ratio> 1 alcoholic liver disease,
myocardial infarction, AST / ALT <1 viral hepatitis
• LD lactate dehydrogenase LD1,2 - myocardial infarction, hemolytic anemia; LD3 - pulmonary embolism; LD4,5 - hepatopathy
diseases of skeletal muscle
• HBD hydroxybutyrátdehydrogenase activity subunit H (LD1,2), myocardial infarction
• GGT gamma-glutamyltransferase hepatopathia (inflammation, alcohol, drugs); test of chronic alcohol consumption;
cholestasis
• ALP isoenzyme of alkaline liver phosphatase - diseases of the biliary tract, bone isoenzyme - bone disease (Paget's disease,
rachitis, tumors), physiologically increased during growth
• ACP acid phosphatase prostatic isoenzyme - prostate tumors, bone isoenzyme - tumor metastasis to bone, osteoporosis
marker
• CK creatine kinase, CK-MB - particularly myocardial infarction, but also in the regeneration of skeletal muscles, chronic
muscular diseases and acute renal failure
• CK-MM - diseases of skeletal muscle, intramuscular injections, physical activity
• AMS amylase (Mr ~ 50,000) pancreatic isoenzyme - acute pancreatitis, salivary isoenzyme – parotiditis
• LPS lipase of acute pancreatitis, acute reversal of chronic pancreatitis
• PSA prostate specific antigen in prostate cancer
• Causes of decreased activity in serum
• CHE cholinesterase chronic hepatopathy, alcoholic-toxic hepatitis (organophosphate intoxication); indicator of hepatic
protein synthesis
TEST
Pathobiochemistry- enzymes and inhibitors 31
Tissue distribution of diagnostically important enzymes
• Tissue damage can be diagnostically prooved indirectly either by determining the activity
of tissue-specific enzymes or by isoenzymes in the blood.
• Tissue-specific enzymes are found preferentially in a particular tissue or have high activity
in that tissue. Exemples of tissue-specific enzymes are listed in the following table.
• Expressionof isoenzymes is mostly determined genetically for each tissue . Therefore,
determination of isoenzymes in the blood enables to identify damaged tissue which they
come from (e.g. pancreatic lipase, CK-MB, LD1).
Organ AST ALT LD LD1 CK GGT ALP ACP AMS LPS CHS
Liver x xx x xxx x xxx
Myocardium x x x xx xx
Muscle x x x xx
Bile duct xx
Kidneys x x x x x
Bones xx x
Erythrocytes x x x xx
Prostate xxx
Pancreas x x xx xxx
Parotid
gland
xx
Pathobiochemistry- enzymes and inhibitors 32