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Chapter 5

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Ryerson University
CHY 204
Mario Estable

CHAPTER 5: PROTEIN FUNCTION Protein Function -Proteins are dynamic molecules whose functions depend on interactions b/w molecules which are affected in physiologically important ways by changes in protein conformations. -The functions of many proteins involve the reversible binding of other molecules; a ligand is a molecule bound reversibly to a protein which can be any molecule incl another pro. A binding site is the site on the pro where the ligand binds which is complementary to the ligand in size, shape, charge & hydrophobic/hydrophilic character. -Proteins are flexible; changes in conformation may be subtle, reflecting molec vibrations & small movements of aa residues throughout the pro, ie it breathes. Induced fit is the structural adaptation that occurs b/w pro & ligand to permit tighter binding. -Enzymes catalyze reactions; substrates are molecules acted upon by an enzyme, ie an enzyme’s ligand. The catalyctic or active site is the ligand binding site for a substrate on an enzyme. 5.1 Reversible binding of a pro to ligand: O2 binding pro -Myoglobin & hemoglobin may be the most studied & best understood pro. There are the 1 pro for which 3D structures were determined & illustrate ligand-pro interaction. -Oxygen can bind to a heme prosthetic group: O2 is poorly soluble in aq solns & can’t be carried to tissues in suff amts if simply dissolved in serum, diffusion is ineffective over dist of few mm & none off the aa side chains in pro are suited for reversible binding of O2 molec. Instead transition metals in them like Cu & Fe have strong tendency to bind O2; free Fe promotes formation of hydroxyl radicals that can damage DNA & other molecules thus are found in forms that sequester it/make it less reactive. Fe is often incorporated into a protein-bound prosthetic group called heme. It consists of a complex organic ring structure called protophyrin w/ Fe2+ bound to it. Fe has 6 coordination bonds, 4 to N that are part of the flat porphyrin ring & 2 perpendicular to prophyrin. The N atoms which have an e-donating character help prevent the conversion of Fe2+ to Fe3+ which doesn’t bind to O2 reversibly. -In heme-containing pro, simultaneous rxn w/ 1 O2 w/ 2 free heme molecules or 2 free Fe2+ that converts Fe2+ to Fe3+ is restricted; 1 of the 2 coordination bonds is occupied by histidine & other is the binding site for O2. When O2 binds, electronic properties of the heme iron changes which accts for the colour change for dark purple to red of O2-rich arterial blood. CO & NO coordinate to heme Fe w/ greater affinity than O2 which is why CO is highly toxic to aerobic organisms. -Myoglobin has a single binding site for O2: Myoglobin is found primarily in muscle tissue, a transport pro, ie. Facilitates O2 diffusion in muscle, abundant in diving animals or those w/ O2 storage function for prolonged times underwater. A single polypep of 153 aa residues w/ 1 molec heme (family of globins which have similar 2° & 3° structures; made up of 8 alpha helical segments connected by bends & ~78% of aa are found in alpha helices. An individual aa reside is designated either by its posn in the aa sequence or by its location in the seq of a particular alpha helical segment. -Pro-ligand interactions can be described quantitatively: function of myoglobin depends on the pro’s ability to bind O2 & release it whenever or wherever its needed. Reversible binding of a pro (P) to ligand (L) can be described by the equilibrium expression: P+L  PL. A higher value of Ka, higher affinity of ligand for the pro. Rate constants, ka & kd are proportionality constants describing the fraction of pool of reactant that reacts in a given amt of time. When the rxn involves 2 molec, eg. P+L…, it’s called 2 nd order & rate constant ka has units m^-1s^-1. when the [ ] of the ligand is greater than [ ] of ligand-binding sites, the binding of the ligand by the pro doesn’t appreciably change the [ ] of free unbound ligand, ie. [L] remains constant. The dissociation constant, Kd, is a reciprocal of Ka (Kd=1/Ka) & in M units; the equilibrium constant for the release fo ligand: (When [L]=K half of the ligand binding sites are occupied, when [L] < Kd, less of the pro has ligand bound to it & in order for 90% of the available ligand-binding sites to be occupied, [L] must be 9x > Kd.) The lower value of Kd, the higher affinity of ligand for the pro. Kd is equal to the M of ligand in which half of the available ligand-binding sites are occupied, ie half-saturation w/ respect to ligand biniding. The more tightly a pro binds to a ligand, the lower [ ] of ligand req for half the binding sites to be occupied & thus the lower value of Kd. Kd = [P][L] / [PL] = kd/ka [PL] = [P][L] / Kd 0 = [L] / [L] + Kd (theta represents the # of binding sites occupied by the ligand) -Can use pressure of O2 (pO2 in kPa) instead of [L], P50 is the partial pressure at which ½ of pro is bound & a lower P50 means higher affinity. -Protein structure affects how ligand binds: specificity (CO binds to free heme molecules 20,000x better than O2, ie. Kd/P50 for CO binding to free heme os more than 20,000x lower than O2 but it binds ~200x better than Ox when heme is bound to myoglobin which may be b/c of steric hindrance: fig 5-5 a) O2 binds to heme w/ O2 axis at an angle, a binding conformation readily accommodated by myoglobin vs. b) CO binds to free heme w/ CO perpendicular to the flat porphyrin ring so when binding to the heme in myoglobin, CO is force to adopt a slight angle b/c the perpendicular arrangement is sterically blocked by His E7/His64, the distal His (His F8=proximal His) & therefore weakens CO- myoglobin bond. The binding of O2 to heme also depends on molec motions/breathing in the pro structure. If the pro were rigid, O2 can’t get in/out easily. -Hemoglobin transports O2 in blood: Normal erythrocytes are 6-9um in diameter & biconcave disks. Are incomplete, vestigial cells that are unable to reproduce & in humans have a 120 day turnover; primary function is to carry Hb which is dissolved in the cytosol at very high [ ] (~34% by weight). Hemocytoblasts are the precursor cells of Hb. In arterial blood, Hb is ~96% sat w/ O2 & in venous blood, ~64% thus each 100mL of blood passing through tissues releases ~1/3 of O2 it carries or 6.5mL of O2 gas at atm pressure & body temp. The Hb tetramer has 4 prosthetic heme groups 2 alpha & beta, similar structure to myoglobin but allows for allosteric binding (binding to one site affects binding to second, ie cooperative binding); involves the Bohr effect. Myoglobin is better for O2 storage having only 1 binding site for O2 vs. Hb is better for O2 delivery having multiple binding sites for O2. -Hb undergoes a structural change in binding O2: The 2 major conformations of Hb are R state (relaxed) & T state (tense). O2 binds to Hb in either state but O2 has sig higher affinity for Hb in R state; O2 binding stabilizes R state & when O2 is absent, T state is more stable which is the predominant conformation of deoxyHb. In tissues, predominant form is deoxyHb & in lungs, oxyHb. Max Perutz propose that the TR transition is triggered by changes in the posns of key aa chains surrounding the heme. In T state, the porphyrin is slightly puckered, causing the hem Fe to protrude somewhat on the proximal His side. The binding of O2 causes the heme to assume a more planar conformation shifting the posn of the proximal His & the attached F helix which lead to adjustments in the ion pairs at alpha1beta2 interface. TR transition also results in narrowing of pocket b/w B subunits. -Hb binds O2 cooperatively: A pro that bound O2 w/ high affinity would bind it eff in the lungs but would not release much of it in the tissues; if the pro bound O2 w/ suff low affinity to release it in the tissues, it wouldn’t pick up O2 in the lungs. Hb solves the problem by undergoing transition from T state (low affinity) to R state (high affinity) as more molec are bound, as a result Hb has a hybrid S-shaped/sigmoid binding curve for O2. b/c of Hb cooperative binding, Hb is more sensitive than Mb to small diff in [O2] b/w tissues & lungs allowing it to bind O2 in the lungs (pO2 is high) & release in tissues (pO2 is low). TR transition occurs more readily in the 2 subunit once O2 is bound to the 1 st subunit; the 4 molec that binds to a heme in a subunit that’s already in R state hence it binds w/ much more affinity than the 1 molec. The Bohr effect states that the affinity of Hb for O2 dec as the [H+] inc & the [CO2] inc (T state); Hb binding of CO2 (15% from tissues) & H+ (40% from tissues) is inversely related to Hb binding to O2. -An allosteric pro is one of which the binding of ligand to 1 site affects the binding properties of another site on the same pro; induced by modulators which interconvert more active & less active forms of pro & either inhibitors.activators. Homotrophic is when the normal ligand & modulator are the same (like in Mb & Hb) vs. heterotrophic is when normal ligand & modulator are diff; some can have 2/more modulators thus can have both homo/heterotrophic interactions. O2 can be considered as both a ligand & activating homotrophic modulator. -Hb also transport H+ & CO2: CO2 + H2O  H+ + HCO3- is catalyzed by carbonic anyhydrase which is particularly abundant in erythrocytes. The binding of O2 by Hb is profoundly influenced by pH & [CO2] so the interconversion of CO2 & bicarbonate is important in regulating O2 binding & release in blood. The binding of H+ & CO2 is inversely related to the binding of O2. At low pH & high [CO2] in peripheral tissues, the affinity of Hb for O2 dec as H+ & CO2 are bound & O2 is released from the tissues. In the lung, as CO2 is excreted & blood pH rises, the affinity of Hb for O2 inc & pro binds more O2 for transport to peripheral tissues. The Bohr effect is the effect of pH & [CO2] on binding & release of O2 by Hb. Hb + O2  HbO2 or more accurately HHb+ + O2  HbO2 + H+ -O2 binds to the Fe atoms of the hemes whereas H+ binds to any of the several aa residues in the pro. The pH of blood is 7.6 in the lungs (R state) & 7.2 in the tissues (R state). -Sickle cell anemia is a molec disease of Hb: over 500 genetic variantss of Hb are known to occur in humans but few are quite rare. The variant genes are called alleles; an individual who has 2 copies of 1 allele is said to be homozygous for that gene vs. if one has 1 copy of each 2 diff alleles, is considered heterozygous for that gene. Sickle cell anemia occurs in individuals who inherit the allele for sickle cell Hb from both parents. When Hb from sickle cell (Hb S) is deoxygenated, it becomes insoluble & forms polymers that aggregate into tubular fibers; normal Hb (Hb A) remains soluble on deoxygenation; insoluble fibers of deoxygenated Hb S causes the deformed, sickle shape of erythrocytes & the proportion of sickled cell inc greatly as blood is deoxygenated. The altered properties of Hb S result from aa substitution from a Glu6 (- charge) to Val6 (no charge) in 2 B chains, ie Hb S has 2 fewer – charges than Hb A. This replacement creates a sticky hydrophobic contact pt at posn 6 of the B chain which is on the outer surface of the molec & thus causes S molec to assoc abnormally w/ each other forming the long, fibrous aggregates characteristic of this disorder. Sickle cell anemia occurs in ppl homozygous for the sickle-cell allele of the gene encoding the B subunit of Hb. Those heterozygous experience sickle-cell trait in which only 1% of erythrocytes become sickled on deoxygenation & have Malaria resistance. 5.2 Complementary interactions b/w pro & ligands: the immune system &
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