Molbiol 2011-11-role of-proteins

Globular Proteins

•  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

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

•  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

•  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.

Structure of heme

porphyrin heme (Fe-protoporphyrin IX)

heme

“distal” histidine

“proximal” histidine

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.

•  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+

O2 Binding in Mb and Hb

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

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

αβ dimer1 αβ dimer 2

T and R forms of Hemoglobin

T = “taut” → deoxy Hb → low affinity for O2

R = “relaxed” → oxy Hb → high affinity for O2

•  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

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

•  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.

•  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

•  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

•  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.

•  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).

•  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

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

•  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.

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.

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

CO2 is bound to hemoglobin at protein interfaces, not Fe2+ center!

•  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.

•  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

•  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

•  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

•  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

Minor Hemoglobins

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.

Globin gene organization

Steps in globin chain synthesis: 1.  Transcription

2.  Modification of mRNA precursor by splicing

3.  Translation by ribosomes & further modifications (i.e. glycosylation)

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

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

Sickle-cell anemia (HbS disease) •  glutamic acid is replaced by valine at position 6 of β chain

normal RBCs sickled RBCs

Symptoms worsen when Hb is in deoxy form - decreased pO2, increased CO2, decreased pH, increased 2,3-BPG

Low solubility of HbS causes aggregation and distortion of cell shape.

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

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

Thalassemias

•  hereditary hemolytic diseases

•  most common genetic disorder in humans

•  heterogeneous collection of diseases

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.

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)

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

Fibrous Proteins

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)

Collagen -‐ most abundant protein in body; rigid, insoluble Elas.n -‐ stretchy, rubber-‐like, lungs, walls of large blood vessels, ligaments

Structure of Collagen

Tropocollagen is a right-‐handed triple helix formed of α-‐chains.

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

Synthesis of collagen

•  made in fibroblast, osteoblasts (bone), chondroblasts (car.lage)

•  secreted into ECM

•  enzyma.cally modified

•  aggregate and are cross-‐linked

Structure of tropocollagen molecule

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.

Biosynthesis of collagen

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.

Vitamin C (ascorbate) deficiency results in scurvy (collagen can’t be cross-‐linked).

Ascorbate coenzyme required by prolyl/lysyl hydroxylase in hydroxyla.on step.

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

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.

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

•  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

Ehlers-‐Danlos Syndrome Hyperextension of skin

Osteogenesis Imperfecta (Blue sclera)

In Utero Radiograph:

•  crumpled long bones

•  beaded ribs

•  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

Elas.n

•  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

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

Enzymes

Enzymes are biological catalysts.

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

Some more nomenclature… Cofactor = inorganic component needed for enzyme

function

Some more nomenclature… Coenzyme = nonprotein small organic component needed for enzyme function

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…

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

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.

1.  Enzyme and substrate combine to form a complex.



2. The complex goes through a transition state – not quite substrate or product


3.  A complex of enzyme and product is produced (EP).

4.  The product is released.

Factors that influence enzyme activity

Environmental factors •  temperature, pH

Cofactors •  metal ions

Effectors •  species that alter enzyme activity

Effect of pH on enzyme activity

Effect of pH on enzyme activity

Examples of optimum pH

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)

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

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]

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

Km is equal to the concentration of substrate required to attain half maximal velocity for any given reaction

Michaelis-Menten Kinetics

•  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

Lineweaver-Burk Plot

Enzyme inhibition

•  many substances can inhibit enzyme activity:

substrate analogs

toxins

drugs

metal complexes

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

Enzyme inhibition

Competitive inhibitor •  resembles the normal substrate and competes for the same site

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

Enzyme inhibition

Noncompetitive inhibitor •  materials that bind at a location other than the normal site •  results in a change in how the enzyme performs

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

Enzyme inhibition

Irreversible inhibitors •  permanently inactivate enzymes

•  heavy metals (Hg2+, Pb2+, Cd2+)

•  aspirin acetylates

•  fluorouracil

•  organophosphates

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

Competitive Inhibition

Uncompetitive Inhibition

Noncompetetive model

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.

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)

Allosteric enzymes show sigmoid curve (cooperative substrate binding like in Hb)

Feedback inhibition

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

ALSO: Troponin T & Troponin I are also released in cardiac damage. Peaks in 8 – 24hr Sensitive and specific for cardiac tissue damage

Molbiol 2011-11-role of-proteins

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