Biochemistry-oral summary exam PDF

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BIOCHEMISTRY - ORAL EXAM1. Globular proteins – myoglobin and hemoglobin. Comparative characteristics of structure and functions. Dissociation curves of myoglobin and hemoglobin. R and T-states. Allosteric properties of hemoglobin. The Bohr effect. Minor hemoglobins, hemoglobinopathies.Globular Prote...

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BIOCHEMISTRY - ORAL EXAM 1. Globular proteins – myoglobin and hemoglobin. Comparative characteristics of structure and functions. Dissociation curves of myoglobin and hemoglobin. R and T-states. Allosteric properties of hemoglobin. The Bohr effect. Minor hemoglobins, hemoglobinopathies. Globular Proteins: Polypeptide chains, which are folded into a spherical or globular shape. Most often contain several types of secondary structure. Myoglobin & Hemoglobin - Heme proteins: Heme proteins comprise a group of specialized proteins with heme as a prosthetic group.

Hemoglobin & Myoglobin comparison: Similarities: - Both bind O2 - Contain heme involved in O2 binding - Almost identical secondary and tertiary structure of Mb and the b-subunit of Hb - Can exert their biological function only if heme contains Fe2+ and not Fe3+ Differences: Myoglobin

Hemoglobin

Number of Heme groups

binds 1 molecule O2 (contains one heme group)

binds 4 molecules O2 (contains 4 heme groups)

Dissociation curve

Hyperbolic

Sigmoid

Affinity for O2

greater affinity for O2 than Hb

lower affinity for O2 than Mb

Structure

Tertiary structure (1 Polypeptide chain)

Quaternary structure (4 Polypeptide chains)

Function

releases O2 for the muscle cells when pO2 → 5mmHg

transports O2 from lungs to tissues where it unloads it

Hemoglobinopathies

1. Structurally abnormal Hb 2. Synthesis of insufficient quantities of normal Hb 3. Combination of both (very rarely)

Increased 2,3-BPG in response to some diseases - O2 Affinity for Hemoglobin is affected by: 1. 2. 3. 4.

Partial O2 pressure (pO2 via “Heme-Heme’’ interactions) [Co2] ⟹ Bohr Effect pH ⟹ Bohr Effect 2,3 Bisphosphoglycerate (BPG)

→ 2,3 BPG decreases Affinity of Hemoglobin for O2 → increased O2 release from Hb to peripheral tissues erhöhte 2,3 BPG level kann durch folgende Krankheiten entstehen: ● Chronic hypoxia (e.g. in case of emphysema) ● Chronic anemia ● High altitude ->circulating hemoglobin may have difficulty receiving sufficient oxygen

2,3-BPG levels are elevated in these diseases, because it lowers the affinity of hemoglobin to O2, permitting a greater unloading of O2 in the capillaries ↑2,3-BPG → ↓ affinity of Hb to О2 → ↑О2 unload on tissues

1. Binding of СО (an Hämoglobin) → The resulting product is carbon monoxy-Hb (carboxy-Hb) → Remaining heme sites bind oxygen with high affinity → Affected Hb exerts higher affinity to О2 and is unable to release it to the tissues (a trap for О2) •Affinity of Hb for СО is 220 times greater than the affinity for О2 • Even minute concentrations of CO can produce toxic concentrations of carbon monoxy-Hb •Binding is tight, BUT REVERSIBLE → Treatment - 100% О2 therapy

What is glycated Hb? What is its diagnostic significance? - HBA1c:

HbA1c ● ↑ blood glucose levels → ↑HbA1c ● HbA1c concentration is used as diagnostic marker for diabetes ● HbA1c represents hyperglycemia state experienced in a period of 2-3 moths before measurement ● Establishment of high HbA1c levels happens in case of prolonged maintenance of high blood sugar levels and diabetes

2. HbS sickle cell disease, HbS disease • point mutation in the β-globin gene → “α2βs2” •Recessive inheritance •Sickle shape of erythrocytes •Shortened lifespan of erythrocytes (~20 days) normal- 120 days, hence the anemia -

macrophages destroy these cells, hence the anemia autosomal recessive disorder, occurs when you have 2 mutant genes

Sickle cell anemia Symptoms: • lifelong episodes of pain • chronic hemolytic anemia • Increased susceptibility to infections HbS – what is the amino acid substitution and how this is related to Hb polymerization and erythrocyte shape? What are the conditions that favor polymerization? Why is this Hb pathologic? :

Amino acid substitution: ● Non-conservative ● hydrophilic (glutamate) is replaced with hydrophobic (valine) → protrusion formation on the β-globin subunit ● The protrusion fits into a complementary site on the α-chain of another Hb molecule in deoxy state → polymerization of Hb at low oxygen tension (↓рО2) ● Deformation of Erythrocytes’s shape

● Erythrocyte destruction (hemolysis) ● Blockage of blood flow in the capillaries and anoxia →pain and death of cells in the vicinity of the blockage

https://www.youtube.com/watch?v=R4-c3hUhhyc factors favoring Hbs polymerization: -

low pO2 (high altitude, flying airplane) high CO2 high H+ high 2,3 BPG -> all factors that favor shift from oxy- to deoxy-Hb and stabilize T-form

Vorteil: - Homo- and heterozygotes are resistant to malaria: → The parasite Plasmodium falciparum (obligatory parasite in erythrocytes) can not complete its intracellular stage of its development

3. Methemoglobin (НbМ) - Oxidation of Fe2+ in heme to Fe3+ (methemoglobin, nonfunctional) - HbM cannot bind O2 → Normally happens very rarely Favoring factors: – Some drugs – Reactive oxygen species – Genetic → Amino acid substitutions in α or β chains may favor HbM formation → NADH methemoglobin reductase (NADH cytochrome b5 reductase) deficiency (the enzyme is involved in regeneration of the reduced state of Hb) In newborns the activity of the enzyme NADH cytochrome b5 reductase is very low!

→ Methemoglobinemia are characterized by “chocolate cyanosis” (a brownish-blue coloration of skin and membranes) and chocolate coloured blood

4. Thalassemias The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. Normally: synthesis of the α- and β-globin chains is coordinated, so that each α-globin chain has a β-globin chain partner. This leads to the formation of α2β2 (HbA). In the thalassemias: the synthesis of either the α- or the β-globin chain is defective or absent Organization of globin genes

β-thalassemias: Reduced or absent synthesis of β-globin chains ● Synthesized α-globin chains can not form stable tetramers → precipitation of α-globin chains → accumulation of γ4 chains (Hb Bart’s) → premature death of cells, initially destined to become mature erythrocytes -

β-thalassemia minor (one defective copy) - > usually don’t need treatment β-thalassemia major (two defective copies)-> healthy at birth, become severely anemic during the first or second year of life ( due to ineffective erythropoiesis), this patients require regular transfusion of blood

α-thalassemias: Reduced or absent synthesis of α-globin chains

● Synthesized chains form tetramers → Accumulation of γ- (Bart’s Hb) and β-globin chains → Premature death of immature erythrocytes β-tetramers: – Show no heme-heme interaction → the dissociation curve is almost hyperbolic representing very high oxygen affinity → useless as О2 deliverers to the tissues (traps for О2) alpha subunits in beta thalassemia can not form tetramers – precipitation of alpha tetramers; beta subunits in alpha thalassemia form nonfunctional tetramers – do not experience heme-heme interactions – can not provide O2 to the tissues Four degrees of the disease: •Silent carrier – one copy is missing •α-thalassemia trait – two copies are missing •HbH - three copies are missing •Hydrops fetalis – death during embryonic development (because α-subunits are needed for HbF synthesis, α2γ2) – four copies are missing

2. Fibrillar proteins – collagen and elastin. Structure, biosynthesis, biologic role.

1. Vitamin C deficiency Vitamin C (Ascorbat) is a cofactor for the hydroxylation of Lysine and proline. This is important, because: - OH-proline => formation of hydrogen bonds, which is important in the stabilization of the triple-helical structure of collagen - OH-Lysine => important for lysyl oxidase, which provides cross-link formation in collagen fibrils Vitamin C deficiency results in: → Insufficient hydroxylation of proline and lysine residues → Decreased collagen fibril strength → Inability to form stable collagen fibrils

Scurvy: - Capillary fragility - Gum and lip bleeding, bruises on the limbs

- Internal bleeding

2. Periodontal disease Periodontal disease is an infectious dental condition, the second most common after caries. The risk group is mainly people over 30 years old, although lately even younger people tend to suffer. Features of the disease are edema and bleeding caused by gingivitis, which can cause bad breath. If not treated, consequences can be recession of gums and outcrop of tooth root.

Main causes for the disease are tartar, unsuccessful dental fillings or prosthesis, avitaminosis, poor dental hygiene, unhealthy lifestyle, misaligned teeth. It is considered that vitamin deficiency is one of the main factors in periodontal progress. Most affected people are pregnant women, patients with diabetes mellitus and malocclusion. Periodontal disease treatment is individual and complex. Primarily the inflammatory processes in the oral cavity have to be suppressed and the tartar has to be eliminated. According to recent studies, supplementation with vitamin C sensibly improves a patient's condition. biological role of Vitamin C in collagen synthesis:

3. Osteogenesis imperfecta (brittle bone disease/ Lobstein syndrome) => Collagen disease

The disease Osteogenesis imperfecta, also known as brittle bone disease or Lobstein syndrome is caused by defects in collagen type 1 synthesis. Common features of the disease are discoloration of the sclera due to abnormal structure of the collagen, usually giving a blue-gray color; bones fracture easily.

→ Dominant mutations in genes for α1 or α2 chains in type I collagen (80% of cases of OI) → Inability to form stable triple helix • Most often the mutation includes incorporation of an amino acid with bulky side chain on the place of glycine (the smallest AA) in the (-Gly-X-Y-) motif • The resulting α-chains prevent proper formation of the triple helical structure → The most severe form (Type II OI): lethal in perinatal period because of pulmonary complications; in utero fractures

3.1 Dentinogenesis imperfecta => collagen disease Another feature is dental disorder known as dentinogenesis imperfecta - disorder of teeth development, dentine abnormality, low mineralization, discoloration/ dentin dysplasia (most often a blue-gray or yellow-brown color), translucent teeth, weak tooth roots. Teeth are also weaker than normal, making them prone to rapid wear, breakage and loss.

4. Ehlers-Danlos syndrome (EDS): => collagen disease •Heterogenic group of disorders – Deficiency in enzymes involved in collagen and fibril processing - N-Procollagen peptidase - cleaves terminal peptides of procollagen - Lysyl hydroxylase (Lysyl → OH-Lysine) substrate for lysyl oxidase, that is important for cross-link formation in collagen fibrils → strength & elasticity. – Amino acid substitutions in genes for collagens I, III (dominant inheritance, vascular form of EDS: potentially lethal because of the risk of arterial rupture); or V (dominant inheritance, classic form of EDS: skin extensibility and fragility, joint hypermotility) ● Collagen that contains mutant chains may have altered: structure, secretion and distribution ● Mutant collagen is frequently degraded ● Incorporation of just one mutant chain may result in degradation of the triple helix.

α1-antitrypsin: biological significance. How mutations in the gene for α1-antitrypsin and smoking are related to elastin stability?

5. Emphysema durch α1-antitrypsin deficiency

Was ist ein Emphysem? : “Bei einem Lungenemphysem sind die Lungenbläschen (Alveolen), an denen der Austausch von Sauerstoff und Kohlendioxid stattfindet, teilweise zerstört und überdehnt, so dass ihre innere Oberfläche verkleinert ist. Ein Lungenemphysem äußert sich durch Atemnot, zunächst nur bei starker Anstrengung, später schon bei leichter Belastung. Ist nicht heilbar.”

-a1- AT is an inhibitor of elastase - elastase is a type of protease ( an enzyme that breaks down other proteins, for example elastin) - elastin is a protein that gives elasticity & strength to lungs. => in case of a1-AT deficiency, a1-AT cannot inactivate elastase, thus elastase breaks down elastin. -if there is an infection or inflammation => neutrophils arise in lungs and make the enzyme Neutrophil elastase ( Neutrophil elastase can break down protein of bacteria AND elastin ) - usually the liver makes a1-AT, which inhibits neutrophil elastase, before it can break down elastin in the lungs. => without a1-AT: - neutrophil elastase damages the walls of the alveoli - without elastin alveoli lose elasticity and structure -acinus turns into one big cavity -> this enlargement of the air spaces is called Emphysema a1-AT deficiency leads to all types of chronic obstructive pulmonary disease or COPD and smoking is another common reason of COPD => if someone with a1-AT- deficiency smokes additionally, then they tend to get earlier onset of COPD than they would have otherwise

Elastin degradation Neutrophil elastase: •Extracellular protease •Destroys elastin in alveolar walls •also destroys structural proteins in other tissues α1-antitripsin (α1-AT): • Secreted by liver, monocytes, alveolar macrophages • Inhibits Neutrophil elastase and other proteases • prevents alveolar walls from destruction α1-AT deficiency

•Destruction of connective tissue of alveolar walls → emphysema -

α1-antitrypsin deficiency leads to increased elastase activity in alveolus

→ Smoking causes oxidation of methionine residue in the active site of the enzyme (α1-AT ) → Enzyme inhibition • Smokers with α1-antitrypsin deficiency are at elevated risk of pulmonary emphysema, than the nonsmokers

3. Glycoproteins and proteoglycans. Structure, biosynthesis, biologic role. Hyalauronic acid is a natural component of connective tissue. It is found in high concentrations especially in the outer layers of the gums, where it has a protective and healing effect on periodontal ligaments. The presence of hyaluronic acid is very important for maintaining gum strength During gum diseases (inflammatory conditions, gum pockets, wounds etc.) the need of hyaluronic acid is elevated (up to 200% compared to basic conditions). Explain the role of hyaluronic acid for building proteoglycans and aggregates. → Proteoglycan monomers associate with a molecule of hyaluronic acid → proteoglycan aggregation It stimulates collagen regeneration. • The association between the core protein and the hyaluronic acid is not covalent – Occurs primary through ionic interactions between the core protein and the hyaluronic acid • The association is stabilized by additional small proteins - link proteins

Enzymes as biocatalysts. Nomenclature and classification of enzymes. Mechanism of enzyme action. Enzyme cofactors. Water-soluble vitamins as enzyme cofactors – cofactors for electron and proton transfer. Water-soluble vitamins as enzyme cofactors for group

transfer.

5. Enzyme kinetics - substrate concentration as a factor affecting enzyme velocity. MIchaelis- Menten plot. Km and Vmax. Linewever-Burk plot. Enzyme concentration as a factor affecting enzyme velocity. Enzyme activity. Effect of pH and temperature on enzyme velocity. --6. Enzyme inhibition. Reversible and non-reversible enzyme inhibition. Kinetics of competitive and non-competitive enzyme inhibition. --7. Allosteric enzymes – structure, kinetics. Regulation of enzyme activity: allosteric regulation, covalent modification, limited proteolysis, induction and repression of enzyme synthesis. ---

8. Isoenzymes – characteristics, biological role. Diagnostic significance of enzymes, functional and non-functional plasma enzymes. Similarities: -

different forms of the one and same enzyme catalyze equal reactions have equal substrate and cofactor specificity some differences are due to differences in the quaternary structure of the enzyme ( for example : lactate dehydrogenase exists in 5 isozyme forms)

Differences: -

amino acid sequence (chemical, physical properties) Regulation (different activator/ inhiitor) cellular, tissue localization Km for substrate and/ or cofactor - LDH1 - isoenzyme for heart muscle,erythrocytes and kidney disease - LDH2- for heart, erythrocyte, kidney and lung disease - LDH3- for lung, thrombocyte and lymph disease - LDH4 - different organs - LDH5 - skeletal muscle, liver

Usage as diagnostic value: Isoenzyme forms from different tissues (creatine kinase, LDH)In case of tissue damage → hypoenzymia in respective tissue (Synthesized in specific tissues (e.g. liver) and secreted in plasma where is the place of their physiologic action) Enzyme marker : I => BB- Brain and gastrointestinal tumor II => MB => heart muscle ( is an indicator of myocardial infarction) II => MM => skeletal muscle Examples of isoenzymes used in clinical diagnostics of different diseases. Pathological condition

Enzymes - diagnostic markers

Myocardial infarction

Creatine kinase 2 (MB isoform) Lactate Dehydrogenase 1 + 2,

Liver diseases

Alanine Transaminase (ALT) Aspartate Transaminase (AST)

Bone diseases

Alkaline Phosphatase (ALP)

→ If these Enzymes are increased in plasma, we can guess which tissue was damaged. “non-functional plasma Enzymes” (except for plasma enzymes: If these enzymes are decreased , we can guess a damage)

Workbook S.13: The Enzyme carbonic anhydrase has a variety of isoenzymes classified in 5 families (alpha, beta, gamma, sigma and epsilon). In humans the common form is the a-type. This enzyme plays an important role in maintaining acid-alkaline balance in kidneys, muriatic acid synthesis in gastric mucosa, ventricular fluid production. Carbonic anhydrase also maintains the pH in the saliva. There are 2 isoforms of the enzyme in the saliva - II and IV. Carbonic Anhydrase IV has carioprotective effect: It attaches to the dental pellicle of tooth enamel and catalyzes the process of binding of H+-ions secreted from the microorganisms. The main biological role of this enzyme is associated with the Bohr-effect. Explain the Bohr-effect and carbonic anhydrase’s role in the lung capillaries and the capillaries of the tissues:

Noch aus dem workbook: S.24 A. 7 (Melika) Methotrexate is a widely used anticancer therapeutics. It is applied to treat leukemia, because it inhibits the reduction of the folic acid (folate) to dihydrofolate (FAH2) and to tetrahydrofolate (FHA4). FHA4 is needed in thymidine monophosphate synthesis. Thus, methotrexate can limit the

leukemia development by inhibiting the DNA synthesis and by suppressing the cell division and proliferation. Explain the biochemical mechanism by which methotrexate can affect the cancer cells ? Usually folic acid is transformed to dihydrofolate (DHF) and dihydrofolate to tetrahydrofolate(FAH4) by the enzyme dihydrofolate reductase (DHFR). Methotrexate inhibits DHFR, so FAH4 is not formed and DNA can not be synthesized. This works in S- phase of the cell cycle, where DNA replication happens

9. Exergonic and endergonic processes. Coupling of exergonic and endergonic processes. High and low energy redox-compounds with biological significance. The central role of ATP in cellular bioenergetics. ATP: energy carrier G: Free energy, required to do work ΔG: represents change in free energy

ΔG = ΔH - T ΔS

ΔH: change in enthalpy( heat released or absorbed during a reaction) ΔS: change in entropy (measure of randomness of the system) Enthalpy & Entrophy= predicts direction of reaction T: absolute temperature ΔG⌃0: represents change in free energy at product/ reactant Exergonic and endergonic processes - ΔG predicts direction of reaction Negative: - G of the system decreases during the rea...