2. Pathobiochemistry
(MB)
2.1. Causes and kinds of disorders. Hereditary metabolic disorders (HMD).
2.2. Enzymes, regulation of metabolism.
2.3. Causes of increased activity of cellular enzymes in plasma. Clinically important enzymes.
Hereditary metabolic disorders
usually AR, GR
usually enzyme
substrate I
product
clinically variable
2.1. Causes and kinds of disorders. Hereditary metabolic disorders (HMD).
• Before: Inborn errors of metabolism
• Definition: diverse group of diseases whose common characteristic is a presence of genetically conditional protein change
• Beginning of 20. century - conception of HMD was formulated - sir Archibald Garrod - 4 HMD
•Today-HMD-more than 700 units
Sir Archibald Edward Garrod,
Sir Archibald Edward Garrod, (25 November 1857 - 28 March 1936) was an English physician who pioneered the field of inborn errors of metabolism. He also discovered alkaptonuria, understanding its inheritance. He served as Regius Professor of Medicine at the University of Oxford from 1920 to 1927.[2]
History
Beginnings of a discovery of HMD are connected with name Archibald Garrod, who pointed to a connection between human diseases and Mendel's principles of a heredity and formulated a concept of HMD (inborn errors of metabolism). Garrod engaged by a study of alkaptonuria and in 1902 published a book The Incidence of Alkaptonuria: a Study in Chemical Individuality, which is first record of human recessive hereditary. In 1923 next his book Inborn Errors of Metabolism was published, where we can find studies about alkaptonuria, cystinuria, pentosuria and albinism.
Pathogenesis of HMD
• HMD are diseases which arise on a molecular level
• Causes of HMD is a change of genetic information (gene,DNA)->bad transcription into mRNA->bad synthesis of protein->protein with a changed structure
• Mutation-^defective transcription-^defective translation
• 1 gene encodes synhtesis of 1 protein molecule
Kinds of mutations: deletion, insertion, lost of a part or whole chromosome
Function of protein in intermediate metabolism
• Enzyme
•Transport protein •Structural protein
• Regulatory protein
Most often-protein works like enzyme
enzyme Substrate -> product
Example p53
• Impact of mutation of a protein function
• Lost of a function
• Amplification of a function - some of protein functions or intensity of a protein production amplificates by a mutation/accumulation
• Profit of a new function
• incorrect protein expression of (in a place and in time)
Accumulation of a substrate (small molecules-for example phenylalanine are difussaly scattered in body fluids, transfered across a filtering barrier of kidneys, excreted by urine. Big molecules-for example mucopolysaccharides accumulate in a place where they arise).
Example = PKU (phenylketonuria) - a mutation of a gene for PAH ( phenylalaninehydroxylase), enzymatic activity < 1% (2 alleles are affected). Low percentage of PKU is caused by a mutation of 1 allele or in a gene for a cofactor of PAH - tetrahydrobiopterin (milder form of PKU).
Lack of a product
Accumulation of a defective enzyme
Synthesis of an incorrect product - block of a metabolic pathway
Lost of various enzymatic activities
Incidence of HMD
• Individual incidence is relatively rare (1:15 000-200 000)
• Collective incidence is high (1:1000)
Incidence of HMD
•Neonatal (newborn) screening 1:1000-1:4000 •selective screening at least 1:500-1:1000
• Frequency of heterozygots for HMD at least 1:15
• Representation differs according a population
• Higher incidence in inbred populations (PKU Turkey, organic aciduria Middle East)
• tyrosinemia l.type Quebec
• aspartylglykosaminuria Finland
• Lysosomal diseases Israel
incidence of HMD in CR
beta-oxidation and OAU
aniniacids
without HPA 18%
HPA and
PKU
13%
saccharides sacharidy
purmy) purines/pyrimidines
mitochondrial
ZU7o
peroxisomal 4%
lysosomal
I07o
CR , 2005, n=127
incidence for CR ~ 1:1000 still -150 various nosologically units
Ways of a HMD transfer
NUCLEAR DNA •Autosomal recessive •Autosomal dominant •Gonosomal dominant •Gonosomal recessive
EXTRANUCLEAR DNA • Maternal type of a heredity (mitochondrial DNA)
Heredity AR (Autosomal recessive)
• The vast majority HMD - for example PKU
• The didease manifests only in a homozygot (it carries both defective alleles for the feature)
• Heterozygot is a clinically healthy individual, it carries a defective gene.
Heredity GR (Gonosomall recessive)
•An abnormal gene of a recessive type is bound to sexual chromosome X
•Clinically it manifests only in men (they have one X chromosome, women have XX)
•When one of parents is affects:
- men are healthy or have the disease
- women can be from 50% carriers
Examples: Hunter's mucopolysaccharidosis, glycogenosis of VIII type
Graphic ilustration of AR and G
R
type of a heredity
mother carrier father carrier
handiccaped chi\d child normal child carrier       carrier child
SCID - Severe Combined Immunodeficiency Disease Autosomal recessive heredity
mother carrier healthy father
J woman handiccaped healthy healthy I carrier man woman man
Gonosomal recessive heredity
Maternal type of a heredity
• Although mitochondrial DNA (mtDNA) has a negligible volume unlike nuclear DNA, the mutations in mtDNA can cause severe diseases.
* 1) Every individual inherits all mitochondrias only after the mother ( mitochondrias of zygote come from the egg, all mitochondrias of sperm vanish).
2) In every cell is about 1000 mitochondrias -1 mitochondria with mutate mtDNA has no impact to the cell. If the mutation in mtDNA manifests in a cellular level or in a whole organism depends on how many percent of mitochondrias have mutate genetic information.
Classification of HMD
1. According a speed of appearing of clinical signs
2. According of individual metabolic systems
3. Acccording a subcellular localization of changed protein
4. According an analytical methodics which are used for an evidence of HMD
l.According a speed of appearing of clinical signs - diseases:
•Akute metabolic
•With an intermitent development •Chronically progressive
2. According of individual metabolic systems - disorders of metabolism
•aminoacids
• carbohydrates
• lipids
• purines and pyrimidines
• High molecular weithgh compounds
• pigments etc.
3. Acccording a subcellular localization of changed protein - HMD:
* cytosolic
* mitochondrial
* lysosomal
* peroxisomal •Golgi apparatus •iont channels etc.
Clinic of HMD
•Exhibitions of DPM - in all age from the birth to an adulthood
* Manifestation - varied, from mild chronically developing forms to acute life threatening states
•A seriousness depends on a level of a disability of changed protein (for example an activity of enzyme 0-20%)
Clinical signs of HMD
•Non-specific- most ( PMR, disorders of muscle tension, disorders of behaviour, disorders of consciousness, convulsions, failure to thrive, vomiting, disorders of heart functions, muscles, liver, kidneys...
•Specific - for example typical abnormal bad smell of urine, sweat...,ectopia of a lens and tromboembolic incidents
Laboratory non-specific discoveries
* Acidosis (for example lactic when it is a deficit of PDH)
* Alkalosis (for example deficit of OTC)
* Hypoglycaemia
* Hyperammonemia
* Hypoketosis (with hypoglycaemia - disorders of (3-oxidation)
* Hyperketosis (some org. aciduria)
* Hypouricemia/hyperuricemia(disorder of met. purines)
* Hypocholesterolemia/hypercholesterolemia (deficit 7-dehydrocholesterol - Smith-Lemli-Opitz sy)
1. Acute metabolic diseases
• Beggining: usually in early neonatal or early infant period
•Signs: respiratoryfailure, sepsis, convulsions, disorders of consciousness, protracted jaundice, development of RDS ci DIC etc.
• Examples: disorders of metabolism of AMK, galactose, ureagenesis, organic acids, (3-oxidation of fatty acids
2. Metabolic diseases with chronical development
•Characteristic: rotation of asymptomatic period with attacks, which typically appear after a load for example a change of nutrition (protein load), febrile period(increased energetic need of organism in developnent of catabolism)...
• Examples: late forms of OTC deficit, some disorders of (3- oxidation of fatty acids
3. Chronically progressive metabolic diseases
•Characteristic: at the beginning normal psychomotoric development stops after the definite period, alternatively a regression comes
• Examples: storage diseases (mucopolysaccharidosis, neurodegenerative diseases...)
Examples of the most famous HMD
• Disorders of metabolism of aminoacids
• Organic acidurias
• Disorders of metabolism of saccharids
• Disorders of metabolism of lipoproteins
• Disorders of metabolism ofpurines and pyrimidines
• Disorders of metabolism of vysocomol. compounds
Laboratoroty diagnostics of HMD
1. In a level of metabolites
2. In a level of enzymes
3. In a molecular level
Diagnosis HMD
substrate
DNA/RNA
Enzymology
Metabolites
Symptoms
Specific - for example:, odor
urine color Nonspecific - for example: coma PMR
dysmorphia hepatho/myopathies and so on
precursor
substrate »o^> By-product
product
• cystine in cystinosis
• cystine in cystinuria
• mucopolysaccharides
Examples:
• glucose in GSD
• ketone bodies in beta-oxidation disorders of fatty acids
• plasmalogenes in peroxisomal disorders
• cysteine in deficiency of CBS
• AdoMet in RM
• ATP in mitochondrial diseases
l.Diagnostics in a level of metabolites
•Characteristic: we prove a changed concentration of a metabolite( substrate, product, abnormal metabolit). The oldest simplest and most spread.
• Utilization: where an enzyme or a transport protein is a defective protein -> in a place of metabolic block a substrate accumulates and a product misses, alternatively other metabolites are synthesized consequantly an activation of alternative metabolic pathways
•Material: serum or plasma, urine, liquor, whole blood in the form of dried blood spots on a filtering paper
I
Diagnosis HMD
Laboratory diagnosis HMD - on several levels:
• Prenatal diagnosis - examination to determine whether the fetus is affected by the HMD, which was demonstrated in the family - only justified cases (AFP, defect in the family)
* Postnatal diagnosis - neonatal screening (PKU hypothyreosis etc.)
Most of the HMD can be diagnosed prenatally - analyzing the enzyme activity or mutation in chorionic villi or amniocytes or by investigation of metabolites in amniotic
fluid.
• Early diagnosis - treatment, compensation
1. Diagnosis at the level of metabolites - continuing
• Investigated metabolites: amino acids, carbohydrates, oligosaccharides, glycosaminoglycans, purines, pyrymidines,lipids, steroids etc.
• Used laboratory techniques: chromatography - paper
- thin layer
- liquid (ion-exchange, high-performance HPLC)
- gas (mass spectrometry GC/MS )
electromigration techniques
- electrophoresis
- capillary electrophoresis
tandem mass spectrometry MS/MS
Presymptomatic diagnosis
substrate
I    D I
examination related risk HMD prenatal diagnosis
screening population segment
one disease or group o diseases known in advanc
neonatal screening
Neonatal screening (NS) = active nationwide search the disease in its preclinical stage.
The analysis of dried blood in filter paper collected by standard procedures from the footer of all neonates.
Hyperphenylalaninemia/ phenylketonuria
• Characteristics: insufficient conversion of Phe to Tyr
• Cause:
1) Deficiency phenylalaninhydroxylase
2) Disorder of the coenzyme tetrahydrobiopterin metabolism
Occurrence: about 1:10 000, the most common DPM
Neonatal screening- in Czech republic from 1975 nationwide - Guthrieho test
Blood collection from neonata screening
Classical criterion for screening
•generally recognized screening test
•credibility of the scr. test: cut-off, fal.neg. Load the healthy population: recally, fal.poz.
•the company is able to secure NS and aftercare of patients retained the organization and economic
Classical criterion for screening
• Diagnostic costs and treatment should be economically balanced in the health care system
• NS is a positive contribution towards the cost of „ benefit/cost"
• NS is a continuous process - efficiency must be constantly evaluated
• screening = method for detecting early forms of disease or deviations from the norm in a given population through test
•is performed on all neonates born in the Czech republic
• rapid diagnosis and early treatment mainly inherited metabolic disorders
• confirm / refute the disease before its symptoms and damage to child
Method of sampling drops of blood from the footer to the neonatal screening card
1962 - founder - prof. Robert Guthrie - introduced a bacterial test for the early detection and PKU and hyperphenylalaninemia in USA (using the strain of the
Bacillus subtilis, they proliferate in the environment of high concentration of phenylalanine)
• from 1969 Guthrieho method in Czech republic, allover screening up from 1975
• from 1985 - the extension of the screening test for congenital hypothyroidism (CH) - iodines deficit fetus, severe damage to the developing brain of a child
• from 2006 - increasing testing of congenital adrenal hyperplasia
(CAH) - previously called adrenogenital syndrome
• 2009 - changes and extensions of screening (according to the Bulletin of the Ministry of Health)
- screening is expanded to include of the screening cystic fibrosis
2. Diagnostics at the level of enzymes
• Characteristics: show reduced activity of the affected enzymes. Testing is difficult (economically costly, often greater burden for the patient -material removal).
• Use: in prenatal diagnosis, to confirm the appropriate DPM, normally precedes testing at the level of metabolites
•Material: leukocytes, erythrocytes and trombocytes isolated from peripheral blood, serum or plasma, culture of skin fibroblast, tissue from muscle or liver biopsy
3. Diagnostics on the molecular level
•Characteristics: diagnosis at the DNA level shows you the defective gene. Economically costly, indicate wisely
• Use: to definitively confirm the diagnosis, where it can be clearly do so on the basis testing of metabolites or enzymes, followed by genetic consulting
•Materiál: leukocytes from peripheral blood, cells from amniotic fluid obtained by amniocentesis, chorionic villus cells obtained by biopsy of the placenta
Symptomatic diagnosis
DNA/RNA	Enzymology	Metabolites	Symptoms
screening	leuko/lymfo	analyte	specific
scanning	fibro	profile of analytes	nonspecific
sequencing	muscle	urine/blood/liquor	coma
dysmorphia hepato/myopathies
The clinical picture of HMD - bodies
A MUSCULAR SYSTEM
The muscular system consists of layers of muscles that cover the bones of the skeleton, extend across joints, and can contract and relax to produce movement.
A SKUUAL SYSTEM
The skeleton is a strong yet flexible framework of bones and connective tissue. It provides support for the body and protection for many of its internal parts.
A CIRCULATORY SYSTEM
This system consists of the heart and a network of vessels that carry blood. It supplies oxygen and nutrients to the body's cells and removes waste products.
A NERVOUS SYSTtM
The nervous system is the body's mam control system. It consists of the bram. the spinal cord, and a network of nerves that extend out to the rest of the body.
A LYMPHATIC (IMMUNE) SYSTEM
This system is a network of vessels that collects fluid from tissues and returns it to the blood. It also contains groups of cells that protect the body against infection
11 MAI I
A RESPIRATORY SYSTEM
The respiratory system is centered on the lungs, which work to get lifc-givtng oxygen into the blood They also rid the body of a waste product, carbon dioxide.
A ENDOCRINE SYSTEM
Many body processes, such as growth and energy production, are directed by hormones. These chemicals are released by the glands of the endocrine system
A DIG! STIVE SYSTEM
The digestive system takes in the food the body needs to fuel its activities It breaks the food down into units called nutrients and absorbs the nutrients into the blood.
A EXCRETORY SYSTEM
The body's cells produce waste products, many of which are eliminated in urine The job of the urinary system is to make urine and expel it from the body
A REPRODUCTIVE SYSTEM
The male and female parts of the reproductive system produce the sperm and eggs needed to create a new person They also bring these tiny cells together.
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The basic situation of the differentia diagnosis of HMDT
•Small molecules
• acutely ill newborn baby
• (repeated) prolonged unconsciousness attack
• failure to thrive infants
• hypoglycaemia
• Large molecules
• progressive disabilities of CNS and muscle
• facial dysmorphia
• organomegaly (liver, spleen, heart)
Abnormal smell and color of urine
smell (small volatile molecules):
• sweaty feet - isovalerate
• caramel/maple syrup - oxoacids
• cooked cabbage - methionine oxide
• fish smell - trimethylamine
• black currant - some organic acids
• mouse smell - phenylacetate
coloring
Common laboratory findings in HMD
Urine
• ketones
• urine acid
• crystalluria
• myoglobinuria
HMD-diagnostic of metabolites
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Sensitivity of methods
• Alkaptonuria: 1-5 g homogentisate /day
• Cystinuria: 1-5 g cystine/day ^^^^^^^^^^^^^^^
Urine - liters for analysis
• Phenylketonuria: 0.1 g phenylalanine /I of blood
• MCAD: C8 acylcarnitine 0.0001 g / I of blood
0.2 - 1 ml serum
Blood paper about 0.05 ml of blood
Amino acids - citrulinemia
MPS I - Hurler disease (deficiency of a -iduronidase)
MPS I in a 6-year-old girl
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Electrophoresis of urinary GAGs
(excretion of dermatan
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Glycoproteinosas - HPTLC oligosaccharides in urine
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Diagnostics of HMD
Principles od enzymatology examination
•Separation of substrate and product •Quantification of gain or loss
substrate
cofactor changed
cofactor •Quantification of gain or loss
Assessment of enzymes in HMD
•Cells are usually necessary
• Leukocytes, fibroblasts
• Fetal tissues and fetal (germ) layers
• Fluorimetric and radiometric techniques (eventually fotometric)
• Measured parameter: the loss of substrate or the product formation
UHMD: 46 enzymes
Typical results of enzymology
Afflicted homozygotes clearly deficient
Heterozygotes: overlay
Healthy homozygotes: usually normal distribution of activity in population
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40
20
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HITEROZYGOTI
Diagnostics of HMD
Substrate
product
OTC: Mutation c. 829 O T (R277W)
A G A A A A A GT/CGG CTCCAGGCT
The normal sequence
AGC  C(AGGGOC   C A
G
G GGAAAGAC GGC
Heterozygous deletion
C COGGGGC
C R
R K t G GMRGACWJRC
Treatment of HMD
Treatment od HMD
1. At the metabolite level
2. At the enzymatic level
3. At the cell level
• The only causal treatment- at the cell level.
• Symptomatic and supportive treatment-mitigates syntomps, not removing the cause.
1. Treatment at the metabolite level
a) Restriction of the gain or the formation of toxic metabolites (eg. diet in PKU, galaktosemia, prevention of catabolism in aminoacidopathies, organic aciduries)
b) Removal of toxic metabolites(peritoneal dialysis, hemodialysis, exchange transfusion) and the use of alternative metabolic pathways(eg. benzoate administration in hyperammonemia)
c) Administration of metabolic inhibitors( eg. allopurinol in hyperuricemia)
d) Replacement of deficient products( eg. arginine in disorders of the urea cycle, tyrosine in PKU)
2. Treatment at the enzymatic level
a) Activation of enzyme by coenzymes delivery at pharmacological doses( eg. pyridoxine in deficiency of cystathionine (3-synazy)
b) Delivery of the deficient enzyme directly - enzyme (eg. in Gaucher and Fabry disease, some types mukopolysachyridoses or glycogenoses)
3. Treatment at the cell level
•Gene therapy with viral or non-viral vectors (yet with no DPM is not used routinely, has its pitfalls
•A special place in the treatment takes transplantation of organs and tissues (eg. liver in tyrosinemia , kidney in cystinosis, bone marrow in adrenaleukodystrofia)
Treatment 1- causa
Treatment 2- influence the path
food
toxic precursor
substrate«
toxic by-product
non-toxic product
V
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Treatment 3- systematic
Elimination of toxins
hemodialysis Hemadsorption Exchange transfusion
General treatment
Energy
Hydration
Treatment of infections Etc.
ENZYMES
Enzymes - proteins - speed up biochemical reactions
1 molecule is transformed noncatalyzed in comparison tol06-1014 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)
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
Binding of the enzyme-substrate
Enzyme-substrate complex
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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
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
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
Mechanisms of regulation of the enzyme activity - 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
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.
(a) Normal binding
(b) Competitive inhibition
(c) Noncompetitive inhibition
Substrate
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Noncompetitive inhibitor
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Exaplex 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. Noncompetitive inhibitor reduces Km and Vmax, as well.
Irreversible 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
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
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.
Induction of B-lactamases are altered so called B-lactam antibiotics :
^penicillins, cephalosporins, carbapenems
A similar effect may have the induction of:
^acetyltransferase, fosfotransferase or nukleotidyltransferase on aminoglykosides
>acetytransferase on chloramphenicol
Other regulation mechanisms of enzyme activity
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
Substrate
Active site   Enzyme Distorted active site v
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Allosteric site __.
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(a) Allosteric inhibition inhibitor
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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
Pleads to change in the conformation of the active site that can bing the substrate in this form.
Examples:chymotrypsinogen —> chymotrypsin prothrombin    thrombin chymotrypsin
MrM^ + Hz° —* ^ V + H3N^
OH' OR'
Polypeptide Poypepiide fragments
R = Phe. Trp. and Tyr; R' * Pro
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.
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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 HCI. 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.
Classification and nomenclature of enzymes
Classes of enzymes:
1. oxidoreduktases
2. transferases
3. hydrolases 4.lyases
5. isomerases
6. ligases
The ending of enzyme names: substrate + -ase
e.g. lactate dehydrogenase, amylase, alcoh dehydrogenase, aspartate transaminase.
The causes of increased activity, enzyme concentration in plasma
1. Patological    cone, 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 degradations Increased phospholipase activity, the degradation of
cytoplasmic membrane phospholipids -> perforations 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 'h enzyme activity (liver) - ALP, GMT etc.
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 hepatoctü -> release into blood.
E.g. Food intakes 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 lg-3 weaks
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).
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 -glomeruli, filtration
>others (the majority) - inactivated in plasma, removed by cells of the RES via receptor, endocytosis
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_Ha If-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
LDHl(HHHH) 4-5 days
LDH5(MMMM) 10 hours
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
• 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. The administration of these drugs increases the plasma levels of GGT.
• ASTc c ALT
• ALP
• ASTrn GGT
http://o.quizlet.com/i/eHnZCzz2IZUlGLwWW5FTw g_m.jpg
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.
Macroenzymes
• Macroenzymes are complexes formed by enzyme linked immunoglobulin, a lipoprotein, protein, or cell membrane fragments.
• Generally makroenzymes have higher molecular weight and longer half-life in blood. Makroenzyme's presence in serum may affect its analytical determination or cause misinterpretations of results.
• Makroamylasemia 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 makroamylase 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.
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	LDX	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		
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 - hepatopathr diseases of skeletal muscle
• HBD hydroxybutyratdehydrogenase 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
clinically important enzymes
Aminotransferases
>provide the conversion of amino acids and keto acids a-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
Clinically important enzymes
a-amylase, AMS
>synthesized in the pancreas, cleaves a-l,4-glycosidic 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
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 non-specific tissue damage according isoforms - electrophoretically (LDH3 pulmonary embolism, myocardial infarction LDH1,2, hepatopathy + disease koster.svalstva LD4,5).