molbiol 2011-11-role of-proteins
TRANSCRIPT
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Globular Proteins
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• a variety of different kinds of secondary structure
Globular proteins are characterized as generally having:
• spherical shape
• good water solubility
• a catalytic/regulatory/transport role i.e. a dynamic metabolic function
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Globular heme proteins contain heme as prosthetic group.
Functions of globular hemeproteins include:
• electron carriers
• part of enzyme active site
• transport of O2 and CO2- hemoglobin
• storage of O2-myoglobin
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• II. Globular Hemeproteins • Contain heme as prosthe.c group • Role of heme is dependent on environment created by 3D structure of protein
• Heme of cytochrome → electron carrier • Heme of catalase → part of ac.ve site • Heme of Hb and myoglobin → binds O2 reversibly
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• A. Structure of Heme • Complex of Protoporphyrin IX & Fe2+
• Fe2+ bound to 4 Ns, other 2 bonds perpendicular to plane of ring available for bonding
• In Hb, one of these aHached to N terminus of His, other binds O2.
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Structure of heme
porphyrin heme (Fe-protoporphyrin IX)
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heme
“distal” histidine
“proximal” histidine
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B. Structure and func9on of myoglobin
• It is a heme protein present in heart and skeletal muscle
• Reservoir for O2 and carrier of O2 in muscle cell
• Single polypep.de chain similar to polypep.des in Hb
• 1. α-‐helical content: • ~ 80% of pep.de in 8
stretches of α-‐helix Labeled A to H
• Terminated by Pro or β-‐bends and loops stabilized by H bonds and ionic bonds.
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• 2. Loca9on of polar and nonpolar amino acid residues: • Interior made up of hydrophobic amino acids stabilized by
hydrophobic interac.ons • Surface → charged amino acids – form H bonds with water
• 3. Binding of heme group: • Heme in crevice lined with non-‐polar amino acids, except 2
His residues • Proximal his9dine – binds directly to Fe2+ of heme • Distal his9dine stabilizes binding of O2 to Fe2+
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O2 Binding in Mb and Hb
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C. Structure and func9on of hemoglobin
• Found exclusively in RBCs → transports O2
• Hb A – predominant form in adults: 4 polypep.de chains -‐-‐ α2β2
• Each subunit – heme-‐binding pocket similar to myoglobin
• Can transport O2 and CO2 • O2-‐binding proper.es affected by allosteric effectors, unlike myoglobin
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1. Quaternary structure of hemoglobin:
• 2 iden.cal dimers: (αβ)1 and (αβ)2
• dimers held together by hydrophobic interac.ons (on contact surfaces of subunits as well as internally) but ionic and H-‐bonding also exist
• 2 dimers held together by weak polar bonds
• different conforma.on in deoxyHb and oxyHb
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αβ dimer1 αβ dimer 2
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T and R forms of Hemoglobin
T = “taut” → deoxy Hb → low affinity for O2
R = “relaxed” → oxy Hb → high affinity for O2
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• a. T form: “taut” form • deoxy form of Hb • 2 αβ dimers joined by ionic and H-‐bonds • low oxygen-‐affinity form of Hb
• b. R form: • binding of O2 disrupts some ionic and H-‐bonds between αβ dimers
• “relaxed” form • high oxygen-‐affinity form of Hb
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D. Binding of oxygen to myoglobin and
hemoglobin
• D. Binding of oxygen to myoglobin and hemoglobin
• Myoglobin → one heme → binds one O2
• Hb → 4 heme→ binds 4 O2 • Hb binding: degree of satura.on
(Y) from 0 to 100% • 1. Oxygen dissocia9on curve: • plot of Y against PO2 • myoglobin : higher affinity for O2
than Hb • P50 is 1 mm Hg for myoglobin and
26 mm Hg for Hb
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• a. Myoglobin: • O2 dissocia.on curve hyperbolic • This reflects that myo binds single O2 • Mb + O2 MbO2 they exist in equilibrium • Exchange between Hb and Mb, Mb and muscle cells depending on equilibrium
• Mb binds O2 released from Hb, releases when O2 drops. Mb then releases the O2 into the muscle cell. This only happens when there is an O2 demand.
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• b. Hemoglobin: • O2 dissocia.on curve is sigmoidal
• Coopera.ve bind of O2 (increased affinity for Hb with more binding)
• Heme-‐heme interac.on: binding of O2 at one heme increases affinity for O2 at others
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• E. Allosteric effects • Ability of Hb to bind O2 depends on allosteric (“other site”) effectors: – PO2
– pH of environment – PCO2-‐ an inc will cause the inc in unloading of O2. – 2,3-‐disphosphoglycerate availability
• allosteric factors do not affect myoglobin
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• 1. Heme-‐heme interac9ons: • structural changes in one heme group transmiHed to others
• affinity for last O2 ~300X affinity for first O2 • a. Loading and unloading of oxygen: • more O2 can be delivered to .ssues with small changes in PO2
• Graph showing loading and unloading at different par.al pressures of O2. Hb alterna.vely carries O2 and CO2 between lungs and .ssues
• b. Significance of sigmoidal O2-‐dissocia9on curve Compare a hyperbolic curve to a sigmoidal curve
• A sigmoidal curve gives increasing affinity of O2 for Hb with increasing par.al pressure while a hyperbolic curve is a straight line in that range.
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• 2. Binding of CO2: • Most of the CO2 in the blood is transported as bicarbonate:
• CO2 + H2O H2CO3
• H2CO3 HCO3-‐ + H+
• Some CO2 binds to the terminal –NH2 of the α and β chains forming carbaminoHb.
• Binding of CO2 stabilizes the “taut” form of Hb (deoxyHb).
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• 3. Binding of CO: • CO binds reversibly to the Fe2+ the same way that O2 does
• CO + Hb HbCO (carbon monoxy Hb) • Affinity of Hb for CO is 220X affinity for O2
• Binding of CO to Hb increases affinity of remaining sites for O2
• O2 dissocia.on curve shigs to leg (becomes hyperbolic)
• > 60% HbCO fatal • treated with O2 therapy
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4. Bohr Effect:
• Shig of O2 dissocia.on curve to the right with decrease in pH or increase in PCO2
• This translates to a decreased affinity of Hb for O2 under these condi.ons, therefore you unload O2 easier
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• a. Source of the protons that lower the pH: • 2 principle sources of protons:
– lac.c acid produced by anaerobic metabolism in muscles – increased produc.on of CO2 by cell u.liza.on of O2 through respira.on:
• CO2 + H2O H2CO3 H+ + HCO3-‐
– in lungs the equilibrium of this reac.on is towards the leg because CO2 is lost through exhaling
• the decreased affinity of Hb for O2 under the Bohr effect condi.ons results is greater off loading (release) of O2 in the .ssues.
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The Effect of CO2 and H+ on O2 Binding
Bohr Effect:
Increased concentrations of CO2 and H+ promote the release of O2 from hemoglobin in the blood.
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How do CO2 and H+ promote the release of O2 from hemoglobin?
• presence of “salt bridge” in T form
• no ionic interaction in R form
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CO2 is bound to hemoglobin at protein interfaces, not Fe2+ center!
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• Summary reac.on for the Bohr effect: • HbO2 + H+ HbH+ + O2 OxyHb DeoxyHb • Equilibrium shigs to the right when H+ conc. increases (decrease in pH), while it shigs to leg when PO2 increases.
• The protonated forms of the terminal α-‐subunit –NH2 groups and His side-‐chains stabilize the T form (deoxy form) of Hb.
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• 5. Effect of 2, 3-‐bis-‐phosphoglycerate(BPG) on oxygen affinity:
• Important regulator of Hb binding O2
• Most abundant organic phosphate in RBC (conc. ~ = conc. of Hb)
• Synthesized from intermediate of glycolysis
• a. Binding of 2,3-‐BPG to deoxyhemoglobin:
• Binds to deoxyHb stabilizing it • Decreases affinity of Hb for O2
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• b. Binding site of 2,3-‐BPG: • 1 molecule of 2,3-‐BPG binds to a
pocket between the β-‐chains in the center of the deoxyHb center
• expelled on oxida.on of Hb (pocket disappears)
• c. ShiX of oxygen-‐dissocia9on curve:
• Blood stripped of 2,3-‐BPG has a high affinity for O2
• 2,3-‐BPG shigs the O2-‐dissocia.on curve to the right allowing decreased affinity of Hb for O2 and effec.ve unloading of O2 in .ssues
• similar to Bohr effect but no difference between lungs and .ssues
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• d. Response of 2,3-‐BPG levels to chronic hypoxia or anemia:
• 2,3-‐BPG increases in chronic hypoxia • chronic hypoxia can be caused by
– pulmonary emphysema or – high al.tudes or – chronic anemia
• increased 2,3-‐BPG shigs O2 dissocia.on further to right allowing greater unloading of O2
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• e. Role of 2,3-‐BPG in transfused blood: • 2,3-‐BPG essen.al for normal transport func.on of blood
• Without normal concs. of 2,3-‐BPG, Hb becomes an O2 trap (doesn’t unload; high affinity)
• Blood for transfusion formerly stored in acid-‐citrate-‐dextrose medium decreased 2,3-‐BPG conc. → “stripped” blood
• Body restores conc. of 2,3-‐BPG in 24 – 48 h • 2,3-‐BPG can be restored by adding inosin
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Minor Hemoglobins
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Minor Hemoglobins
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Minor Hemoglobins
Embryonic form is Hb Gower 1 (ζ2ε2) (yolk sac).
HbF - 2 α chains, 2 γ chains (β-chain family) - major form in fetus and newborn (fetal liver –2 weeks).
HbA - 2 β chains, 2 α chains - major form in adult.
Fetal bone marrow begins synthesizing HbA around 8th month.
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Globin gene organization
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Steps in globin chain synthesis: 1. Transcription
2. Modification of mRNA precursor by splicing
3. Translation by ribosomes & further modifications (i.e. glycosylation)
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Hemoglobinopathies
• caused by abnormal structure of Hb • characterized by low levels of normal Hb
Sickle-cell anemia (Hemoglobin S disease) Hemoglobin C disease Hemoglobin SC disease Thalassemias – α thalassemia
β thalassemia
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Sickle-cell anemia (HbS disease)
• abnormal β chain. HbS = α2βS2
• β chain mutation - glu 6 à val 6
• glu is negatively charged, val is nonpolar. • only has effect postnatally because HbF is major species in fetus
• symptoms - hemolytic anemia, painful crises, poor circulation, frequent infections
• heterozygotes - HbA and HbS both present - 1 in 10 African Americans; "sickle cell trait" - no symptoms, normal life span
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Sickle-cell anemia (HbS disease) • glutamic acid is replaced by valine at position 6 of β chain
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normal RBCs sickled RBCs
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Symptoms worsen when Hb is in deoxy form - decreased pO2, increased CO2, decreased pH, increased 2,3-BPG
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Low solubility of HbS causes aggregation and distortion of cell shape.
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HbS • val instead of glu at position 6
HbA • glu at position 6
HbC • lys instead of glu at position 6
HbSC • HbS as well as HbC present → 2 bands in electrophoresis
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HbC disease • lys instead of glu at position 6
• HbC homozygotes - mild, chronic hemolytic anemia. Not life- threatening
HbSC disease • HbS as well as HbC present → 2 bands in electrophresis
• usually undiagnosed until infarctive crisis occurs (childbirth, surgery) • can be fatal
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Thalassemias
• hereditary hemolytic diseases
• most common genetic disorder in humans
• heterogeneous collection of diseases
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β-thalassemias • synthesis of β-chain decreased or absent
β-thalassemia minor (or trait) - one normal, one defective β-chain gene. Not life-threatening
β-thalassemia major - both genes defective. Normal at birth.
Severe anemia by age 1-2.
Treatment requires frequent transfusions → Leads to iron overload (hemosiderosis).
Death between 15-25 years old. Bone marrow transplant (BMT) is an option.
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α-thalassemias
• decreased or absent α chain synthesis
• severity of disease depends upon the number of defective α genes:
0 defective - normal
1 defective - silent carrier of α-thalassemia. No symptoms
2 defective - α-thalassemia trait - no serious symptoms
3 defective - Hemoglobin H disease - moderately severe hemolytic anemia
all 4 defective - hydrops fetalis - fetal death (α chains needed for HbF)
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Methemoglobinemia
• 1. Forma9on of methemoglobin • Oxida.on of Fe2+ → Fe3+ converts Hb and myoglobin to metHb and metmyoglobin
• Cannot bind O2, • Oxida.on by drugs like nitrates, H2O2 or free radicals or muta.on in α-‐ or β-‐chain of globin → methemoglobinopathy (HbM).
• a. Reduc9on of methemoglobin: • Normal oxida.on corrected by NADH-‐cytochrome b5-‐reductase
• RBCs of newborns → half the capacity of this enzyme, therefore more suscep.ble to oxida.on
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Fibrous Proteins
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Fibrous proteins are characterized as generally having: • one domina.ng kind of secondary structure (i.e. collagen helix in collagen)
• a long narrow rod-‐like structure
• low water solubility
• a role in determining .ssue/cellular structure and func.on (e.g. collagen, α-kera.n)
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Collagen -‐ most abundant protein in body; rigid, insoluble Elas.n -‐ stretchy, rubber-‐like, lungs, walls of large blood vessels, ligaments
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Structure of Collagen
Tropocollagen is a right-‐handed triple helix formed of α-‐chains.
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The α-‐chains (individual polypep.des composing tropocollagen) consist of -‐[Gly-‐X-‐Y]-‐ repeats. Proline and hydroxyproline/hydroxylysine are ogen present in the X and Y posi.ons, respec.vely.
Structure of Collagen
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Synthesis of collagen
• made in fibroblast, osteoblasts (bone), chondroblasts (car.lage)
• secreted into ECM
• enzyma.cally modified
• aggregate and are cross-‐linked
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Structure of tropocollagen molecule
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Biosynthesis of collagen
1. forma.on of pro-‐α-‐chains -‐ contains signal sequence – promotes binding of polysome to RER and secre.on into the cisternae; signal sequence removed
2. some pro and lys residues (in the Y posi.on of gly-‐X-‐Y) are hydroxylated by prolyl hydroxylase and lysyl hydroxylase; needs molecular O2 and reducing agent like ascorbic acid (from vitamin C).
3. glycosyla.on -‐ glucose and galactose added to hydroxylysines; pro-‐α-‐chains join to form procollagen. N-‐ and C-‐terminal extensions form interchain disulfide bonds; central triple helix formed because of favorable alignment; Transported to Golgi, packaged, and secreted as procollagen.
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Biosynthesis of collagen
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Biosynthesis of collagen (cont’d)
4. N-‐procollagen pep.dase and C-‐procollagen pep.dase remove terminal extensions, leaving triple helical collagen (occurs extracellularly).
5. collagen fibrils -‐ form by associa.on of collagen molecules with about a 3/4 overlap with other molecules (staggered, parallel arrays)
5. cross-‐linking -‐ interchain cross-‐links caused by lysyl oxidase (a pyridoxal phosphate and copper-‐requiring enzyme); O2 required; oxida.ve deamina.on of lysines and hydroxylysines; Allysine (aldehyde) reacts with amino group of nearby lysine or hydroxylysine to form interchain cross-‐link. Very important for tensile strength of collagen.
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Vitamin C (ascorbate) deficiency results in scurvy (collagen can’t be cross-‐linked).
Ascorbate coenzyme required by prolyl/lysyl hydroxylase in hydroxyla.on step.
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Cross links formed by lysyl/prolyl oxidase -‐ copper coenzyme Number of cross-‐links increases with age → causes s.ffening, decreased elas.city of skin and joints.
Cu2+/ vitamin B6
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Biosynthesis of collagen (con’t) In the final step, collagen fibrils form spontaneously from tropocollagen.
covalent X-‐links between Allysine and hydroxylysine
tropocollagen molecule
triple helix of α-‐chains.
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Types of Collagen
Type Common disorders Representative Tissues
I
Ehlers-Danlos Osteogenesis Imperfecta Marfan’s
skin, bone, tendons, cornea
II - cartilage, intervertebral disks, vitreous body
III Ehlers-Danlos blood vessels, lymph nodes, dermis, early phases of wound repair
IV Alport’s Goodpasture’s
basement membranes
X - epiphyseal plates
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• degrada.on of collagen by collagenase allows remodeling of ECM
Collagen Degrada.on and Disorders
Ehlers-‐Danlos – hyperextensive joints, hyperelas.city of skin, aor.c aneurisms, rupture of colon, skin hemmorhages due to muta.on in α-‐chains
Osteogenesis Imperfecta – briHle bone disease, mul.ple fractures, blue sclera, hearing loss, retarded wound healing
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Ehlers-‐Danlos Syndrome Hyperextension of skin
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Osteogenesis Imperfecta (Blue sclera)
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In Utero Radiograph:
• crumpled long bones
• beaded ribs
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• rubber-‐like proper.es
• connec.ve .ssue protein
• lungs, large blood vessels, elas.c ligaments
Composi.on: -‐ small nonpolar amino acids (Gly, Ala, Val) -‐
also rich in Pro and Lys -‐ liHle or no OH-‐Pro or OH-‐Lys
Elas.n
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Elas.n
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• 3D network of cross-‐linked polypep.des
• cross links involve Lys and alLys
• 4 Lys can be cross-‐linked into desmosine
• desmosines account for elas.c proper.es
Elas.n
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Elas.n Degrada.on and Disorders
• in lungs -‐ lung alveolar elas.n in constantly exposed to neutrophil elastase α1-‐AT inhibits elastase thus preven.ng loss of lung elas.city
• individuals who are homozygotes for mutant α1-‐AT are very suscep.ble to emphysema
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Enzymes
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Enzymes are biological catalysts.
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Some nomenclature… Active site = special pocket where substrate binds Specificity 1. enzymes are specific for a single molecule or a structurally related group of substrates 2. usually only 1 enzyme per reaction type
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Some more nomenclature… Cofactor = inorganic component needed for enzyme
function
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Some more nomenclature… Coenzyme = nonprotein small organic component needed for enzyme function
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Holoenzyme - the enzyme protein plus its cofactor
Apoenzyme - enzyme protein without its cofactor
Prosthetic groups – a coenzyme that’s very tightly (usually covalently) attached to the protein, such as heme
Some more nomenclature…
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How Enzymes Work Enzymes increase the rate of reactions without themselves being altered in the process of substrate conversion to product. This defines a catalyst. Enzymes increase reaction rates by lowering the energy input needed to form a reactant complex that will eventually form product. This occurs via the formation of a complex between enzyme and substrate (ES):
E + S ES E + Pk1 k2
k-1
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Steps in an Enzymatic Reaction
1. Enzyme and substrate combine to form a complex.
2. Complex goes through a transition state – not quite substrate or product
3. A complex of the enzyme and the product is produced.
4. Finally, the enzyme and product separate.
All of these steps are equilibria.
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Steps in an Enzymatic Reaction
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1. Enzyme and substrate combine to form a complex.
Steps in an Enzymatic Reaction
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Steps in an Enzymatic Reaction
2. The complex goes through a transition state – not quite substrate or product
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Steps in an Enzymatic Reaction
3. A complex of enzyme and product is produced (EP).
4. The product is released.
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Factors that influence enzyme activity
Environmental factors • temperature, pH
Cofactors • metal ions
Effectors • species that alter enzyme activity
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Effect of pH on enzyme activity
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Effect of pH on enzyme activity
Examples of optimum pH
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Effect of temperature on enzyme activity
• exceeding normal temperature ranges always reduces enzyme reaction rates
• optimum temperature is usually 25 - 40 ºC (but not always)
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Kinetics • Kinetics is the study of the rate of change of reactants to products
• Velocity (v) refers to the change in conc. of substrate or product per unit time
• Rate (k) refers to the change in total quantity (of reactant or product) per unit time
• Initial velocity (v0) is the change in reactant or product conc. during the linear phase of a reaction
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Michaelis-Menten Kinetics Three basic assumptions: 1: ES complex is in a steady state, i.e.
remains constant during the initial phase of a reaction
2: when enzyme is saturated all enzyme is in the form of ES complex 3: if all enzyme in ES then rate of product
formation is maximal:
Vmax = k2[ES]
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The Michaelis-Menten equation is a quantitative description of the relationship between the rate of an enzyme catalyzed reaction (v1), substrate concentration [S], the M-M rate constant (Km) and maximal velocity (Vmax)
Michaelis-Menten Kinetics
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Km is equal to the concentration of substrate required to attain half maximal velocity for any given reaction
Michaelis-Menten Kinetics
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• Lineweaver and Burk manipulated the MM equation by taking its reciprocal values generating a double reciprocal plot
• Leads to a linear graph of the reciprocals of velocity and substrate concentration
Lineweaver-Burk Analysis
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Lineweaver-Burk Plot
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Enzyme inhibition
• many substances can inhibit enzyme activity:
substrate analogs
toxins
drugs
metal complexes
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Enzyme inhibition - 2 broad classes:
Irreversible inhibition • forms covalent or very strong noncovalent bonds • site of attack is amino acid group that participates in the normal enzymatic reaction
Reversible inhibition • forms weak, noncovalent bonds that readily dissociate from an enzyme • the enzyme is only inactive when the inhibitor is present
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Enzyme inhibition
Competitive inhibitor • resembles the normal substrate and competes for the same site
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Enzyme inhibition
Examples of competitive inhibitors: • methanol and ethylene glycol compete with ethanol for the binding sites to alcohol dehydrogenase
• methotrexate competes with folic acid for dihydrofolate reductase
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Enzyme inhibition
Noncompetitive inhibitor • materials that bind at a location other than the normal site • results in a change in how the enzyme performs
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Enzyme inhibition
Examples of noncompetitive inhibitors: • physostigmine is a cholinesterase inhibitor used in the treatment of glaucoma
• captopril is an ACE inhibitor used in treatment of hypertension
• allopurinol is a xanthine oxidase inhibitor used to treat gout
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Enzyme inhibition
Irreversible inhibitors • permanently inactivate enzymes
• heavy metals (Hg2+, Pb2+, Cd2+)
• aspirin acetylates
• fluorouracil
• organophosphates
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Enzyme Inhibition - Summary Competitive • Inhibitor binds at substrate site, inhibition is reversible as higher substrate competes for inhibitor, Vmax unchanged, Km increased
Noncompetitive • Inhibitor binds at site other than substrate, ESI cannot form product, increased substrate does not compete, Km unchanged, Vmax decreased
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Competitive Inhibition
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Uncompetitive Inhibition
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Noncompetetive model
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Enzyme Regulation • Proteolytic cleavage to activate: Enzyme exists in inactive form (zymogen) that is activated by removal of a short peptide segment ( truncation) • Covalent modification to increase or decrease activity, most common is phosphorylation • Sequestration: enzyme forms inactive polymers
• Allosteric (“other site”) regulation, both positive and negative ( homotropic, heterotropic) Induction-upregulation: increase gene expression, synthesis of more enzyme molecules
Repression-downregulation: decrease gene expression, decrease synthesis of enzyme molecules.
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Allosteric enzymes Are regulated by molecules called effectors (modifiers) that bind non-covalently at a site other than active site. They can alter Vmax or Km or both) 1. Homotrophic effectors – when the substrate itself is an effector
2. Heterotrophic effector – when the effector is different from a substrate (often it is an end-product - feedback inhibition)
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Allosteric enzymes show sigmoid curve (cooperative substrate binding like in Hb)
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Feedback inhibition
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Enzymes Used in Clinical diagnoses
Tissue damage: Increased release of tissue enzymes in plasma
Enzyme assay is used for both diagnostic and prognostic purpose Eg: ALT – present in the liver will be appearing in the plasma if there is Liver damage or cell necrosis
Isoenzymes: Structurally different enzymes but catalyze the same reaction Eg: CK1, CK2, CK3 (creatine kinase, CK MB (CK 2) is present in the heart, its presence in plasma is indicative of myocardial infarction
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ALSO: Troponin T & Troponin I are also released in cardiac damage. Peaks in 8 – 24hr Sensitive and specific for cardiac tissue damage