3/3 notes: Hemoglobin and Myoglobin
3/3 notes: Hemoglobin and Myoglobin Chem 349
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This 18 page Class Notes was uploaded by Allie Bartlett on Sunday March 8, 2015. The Class Notes belongs to Chem 349 at a university taught by Dr. Runquist in Fall. Since its upload, it has received 216 views.
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Date Created: 03/08/15
Quiz answers 1 An amphipathic helix is A helical structure that has both a nonpolar and polar surfaces 2 The net charge of the amino acid tyrosine at pH 75 is O a The pKa of Tyrosine is 105 at pH75 the OH is still protonated therefore it does not add any more protons leaving it at neutral zero 3 The net charge of the pentapeptide gt H3Nlysser hisargasnCOOH at pH 75 2 1 1 0 0 1 0 1 4 The PI of lysine is 975 which is the isoelectric point the pH when a molecule has a net charge of 1 5 The hydrogen the that would be involved in stabilizing the helix are In the backbone asppeptideboncserinepeptide bondCO 0 VIP ln Secondary structures Hydrogen bonding is between the backbone o Tertiary structures hydrogen bonding is between 3 groups Hemoglobin can bind 4 oxygen molecules 0 When hemoglobin is the Iron Fe is being residue When the hemoglobin Hb is the Fe is then ulled u into th m the His residue is moved at the 5th connection point has a because it has a Globular Protein H20 Soluble Globular Protein H20 Soluble Ligand 02 Binding Ligand 02 Binding Function Bind amp release 02 to muscle cells Function Take 02 from the lungs to the tissues including muscles Structure 1 polypeptide 8 alphahelices highest level is tertiary only 1 polypeptide chain Structure 4 polypeptide subunitsalpha2 beta2 Quaternary structure Has 1 heme groupprosthetic group Able to bind 4 02 molecules each one has a heme group Polar sidechains are on the surface hydrophobic are on the inside Has 2 histidine Polar side chains 1 Proximal anchors heme group 2 Distal helps bind 02 amp reduced binding affinity of CO carbon monoxide Displays cooperativity once 1 02 binds the affinity for 02 at the other subunits increases Allosteric Regulation Has higher affinity for 02 than hemoglobin bc myoglobin has to grab 02 from the hemoglobin Takes more 02 to get Hb saturated once the 02 binds however the affinity increases E I 31sz M r r L lj l r A A Ham A r v E r H l 3 Ea m l with GEEL FEE mail 0 The is a sigmoidal curve which suggests that there is Which indicates that the first 02 bound is hard but gets easier 0 The I is almost fully saturated at 2040 p02 0 is an allosteric protein I It can bind other ligands other than oxygen which can affect the affinity of binding to oxygen 0 Protons H Tacid 0 Carbon Dioxide C02 0 23bisphosphoglycerate Stripped Hb is a A hemoglobin solution from which endogenous 23 bisphosphoglycerate has been removed 0 Two types of Both contain 2 alpha and 2 beta secondary structures I TENSE Deoxygenated Hb T Contains High 23BPG acidic pH High CO2 which help deoxygenate the Hb It has low oxygen affinity and releases oxygen in the tissues I RELAXED Oxygenated R at basic pH low CO2 and very low conc of 23BPG will better bind with oxygen 23Bisphosphoglycerate is bound to deoxyhemoglobin via ionic interactions It is negatively charged due to the two phosphate groups Figure 713 Binding of 2 arbuisphasphoglayeerate to deoxyhemoglobin From Mathews and van Hulda may 39QquotI39m EartjamIim Cwmmi gE Publishing Inca EdiFE Elquot Hemagilebiii tf g of mam Eim i 1 E PE 3 5 mmoliL E E 39 E mm li L j quot RENEdual E El Hittit39udesiu f A a I i i i i Ii i i am an llE Iili Partial we lei ew gem Elf1m HE o Stripped Hb BLACK allows for higher affinity binding oxygen lower Kd higher affinity y 0 Regular Hb BLUElwith 23BPG gives a lower affinity to bind to 02 and a HIGHER Kd Effects of pH on oxygen binding 0 More BASIC Higher affinity to bind 02 more saturated o reduced affinity to bind 02 less saturated Effect of C02 on Oxygen Binding Reduced affinity to bind oz 0 C02 can bind to Hb amp modify the Nterminus o C02NH2R gt COZNHR Factors Stabilize the T State E DEOXY LOW 02 BINDING HIGH 02 BINDING 23bisphosphoglycerate Contains lots of 23BPG Does not contain much Enhances the ability of RBCs to release oxygen near tissues that need it most thus an allosteric effector Conc of H High acid Low acid Conc of C02 High C02 Low C02 The Bohr Effect Eexhr effect curves iilll 7 an E E 3 E EEI I High 1112 low pH D gh i 39 IInrmal CUE normal pH E E LmIII CUE hlgh pH 2 and Lungs tissueg EIIUEDII I I II I I I II I 391 all E El quot39IIZI DHygen ICII39ESSUI39E mm Hg an increase in blood 002 concentration which leads to a decrease in blood pH will result in hemoglobin proteins releasing their load of oxygen Conversely a decrease in carbon dioxide provokes an increase in pH which results in hemoglobin picking up more oxygen Since carbon dioxide reacts with water to form carbonic acid an increase in 002 results in a decrease in blood pH o In deoxyhemoglobin the Nterminal amino groups of the dsubunits and the Cterminal histidine of the Bsubunits participate in ion pairs The formation of ion pairs causes them to decrease in acidity Thus deoxyhemoglobin binds one proton W for every two 02 released 0 ln oxyhemoglobin these ion pairings are absent and these groups increase in acidity Consequentially a proton is released for every two 02 bound Specifically this reciprocal coupling of protons and oxygen is the Bohr effect2 CUE 2r is tiissuss 1 isslssd 2 in blsstl 2 llb E if syh msgls bin I miss Hljj s 1 HuiHm g 1 Lissa Tissus Hle GE 1 1 HEMi 1 fllll DE a ADE a lLun s 1 issslssd 422 in plasma 2 I iltl39AHllflrEFl Dt l fEdl hsm HCCZI3r hil l j l l f je r e EDE lIED in lungs 3 HEBDD stmsssr sndissidslrIHi prtsnj Bohr cozH Carbon dioxide amp Protons are affecting the affinity of Hb to bind to Oxygen Fetal Hemoglobin o 2 alpha chains 0 2 gamma chains INSTEAD OF BETA O SerineDoes not bind well with 23BPG replaces HistidineTypically found in the cavity and binds 23BPG 0 Because the fetal Hb cannot bind 23BPG very well the affinity to bind 02 increases Ex Sickle Cell RBC s are caused by one basepair change in a Beta chain Hydrophobic AA Valine replaces Glutamate Disrupts the folding of Hb COLLAGEN Structural protein of the extracellular matrix One of the most prevalent and common proteins IMPORTANT EXAM ll Q Collagen is unique because it has unusual structural features three parallel polypeptide strands in a lefthanded polyproline lltype PPII helical conformation coi about each other with a oneresidue stagger to form a righthanded triple helix The tight packing of PPquot helices within the triple helix mandates that every third residue be Gly resulting in a repeating XaaYaaGly sequence The amino acids in the Xaa and Yaa positions of collagen are often 2Sproine Pro 28 and 2S4R4hydroxyproline Hyp 38 respectively ProHypGly is the most common triplet 105 in collagen 9 Rod shaped Enzyme is a protein catalyst Proteins working as enzymes What is an enzyme A Protein that decreases the energy of activation for a reaction Simple enzyme catalyzed rxn ES gtlt ES gtlt PE Enzyme substrate gt ES omple gt Can make the product or go back to substrate 0 The enzyme is unchanged and able to do many reactions 0 Enzymes speed up reactions and take less energy Enzyme properties 1 MUST bind with substrate 2 MUST stabilize the transition state EX Chymotrypsin is a digestive enzyme which cleaves peptides on the carboxy terminal side of hydrophobic amino acids Binds to protein in the active site QHow do enzymes lower the activation energy stabilize the transition state The active site of the enzyme 0 Allows two molecules to interact by decreasing distance btwn them 0 Decreases movement 0 Holds in correct orientation amp proximity 0 Provides specificity shape amp geometry c There is a specificity pocket driven by the active site ACTIVE SITE CAVITY of Chymotrypsin 903 Has a unique microenvironment nonpolar pH The site is three dimensional The site represents a very small region of the enzyme Substrates bind by WEAK INTERACTIONS electrostatic hydrogen bonds van der waals hydrophobic In order to be able to easily release product Chymotrypsin Chymotrypsin is a digestive enzyme belonging to a super family of enzymes called serine proteases It uses an active serine residue to perform hydrolysis on the C terminus of the aromatic amino acids of other proteins Chymotrypsin is a protease enzyme that cleaves on the C terminal phenylalanine F tryptophan W and tyrosine Y on peptide chains It shows specificity for aromatic amino acids because of its hydrophobic pocket httpchemwikiucdaviseduPhysicalChemistryKineticsCaseStudies3AKineticsChymotrypsin Enzymes bind substrates via two methods 1 2 Lock amp Key The substrate has a perfect fit for the enzymes Inducedfit Not only 1 substrate This is the most tailored model Know what first and second order Kinetics are First order If a reaction rate depends on a single reactant and the value of the exponent is one o A first order reaction has a rate proportional to the concentration of one reactant o ratekA or ratekB o First order rate constants have units of sec 1 In other words a first order reaction has a rate law in which the sum of the exponents is equal to 1 Second order when the overall order is two a secondorder reaction has a rate law in which the sum of the exponents is equal to 2 A secondorder reaction has a rate proportional to the product of the concentrations of two reactants orto the square of the concentration of a single reactant For example each of the equations below describe a secondorder reaction ratekA2 RatekB2 RatekAB Kinetics How fast a reaction will happen Think Movementmotion Thermodynamics Will a reaction even have a chance of happening What best describes a first order reaction It is the reaction where the rate of the reaction is directly dependent of the concentration of substrate substance Rate constant skgt P Rate ks1 rutroam Hate Eh Rate S2 b mcrmgg Rate S2 Kinematics allows us to predict reactions MichaelisMenten Equation A rate equation for an enzyme catalyzed reaction IMPORTANT FOR EXAM k1 represents the rate constant k1 k2 A E S 5 ES 5 E P k k 2 Vo is the initial Vo Vmax 1 concentration K m Vmax represents the maximal rate or velocity that the enzyme is able to convert the substrate into product Vmaxenzyme When VELOCITY is at VMAX the ENZYME is SATURATED With substrate MichaelisMenten kinetics V0 varies with S V Vmax Vmax max approached asymptotically V0 is moles ot product tormed per sec when P is low close to zero time max2 quot39 E SltgtESE P l MichaelisMenten Model Reaction velocity V0 g i V0 Vmax Km KM MichaelisMenten Equation Substrate concentration S e 1 As soon as the Enzyme is released in the equation there is so much substrate that it immediately binds back to the enzyme gt gt gt gtEIK2 gtEP rateK2ES When K2 is high the rate of the reaction is high o Michaelis Constant Equation k l k2 E 2a When KMSubstrate Velocity 12 Vmax KM Michaelis constant 0 Indicates how effectively the enzyme selects its substrate and converts it to product 0 A LOW KM enzyme is MORE EFFECTIVE at LOW Substrate 0 KM under certain cases can reflect the Kd of the ES complex 0 KMESES 0 High KM Low ES complex 0 High Kmpoor affinity for a substrate to bind to Enzyme o For a good reaction we want a HIGH Kcat amp LOW KM c To find the Km for an enzyme using the Michaelis Menton plot we first 0 1 Find 12 Max Volume 0 2 Draw the line down to the xaxis which represents the KMSubstrate units is in uM Ex if the Km of an enzyme for substrate x is 1000uM and the Km for substrate Y is 10uM which substrate binds BETTER to the enzyme Answer Substrate Y will bind better By determining the Vmax the rate constant K2 Kcat can be found o VVmaxK2 Et 0 KcatKm Want a HIGH Kcat amp LOW Kmbut not too low 0 The enzymes that are close to kinetically perfect have a KcatKm ratio that are 0 Fumarase o Triose Phosphate isomerase Enzyme Inhibitors 0 Types of REVERSIBLE Inhibitors 0 Competitive Most common Competes with the substrate directly 0 close to th Inhibitors tieup the enzyme at the first rxn or at the complex gt O E S gt ES INHIBITOR Which Impact on Impact on MM Enzyme Km Vmax GraphGrap Form does h Schematic inhibitor bind E E8 or both Competitive E only NA Because the KM is increased the graph shifts to R showing a LOWER affinity Uncompetiti Able to bind 39 ve at E8 Noncompet Able to bind NA 39 itive E amp ES Competitive inhibitors as the name suggests compete with substrates to bind to the enzyme at the same time The inhibitor has an affinity for the active site of an enzyme where the substrate also binds to This type of inhibition can be overcome by increasing the concentrations of substrate outcompeting the inhibitor Competitive inhibitors are often similar in structure to the real substrate Competitive inhibitors can only bind to E and not to ES They increase Km by interfering with the binding of the substrate but they do not affect Vmax because the inhibitor does not change the catalysis in ES because it cannot bind to ES Competitive Inhibitor o Uncompetitive inhibitors bind to the enzyme at the same time as the enzyme39s substrate However the binding of the inhibitor affects the binding of the substrate and viceversa This type of inhibition cannot be overcome but can be reduced by increasing the concentrations of substrate The inhibitor usually follows an allosteric effect where it binds to a different site on the enzyme than the substrate This binding to an allosteric site changes the conformation of the enzyme so that the affinity of the substrate for the active site is reduced Uncompetitive inhibitors are able to bind to both E and ES but their affinities for these two forms of the enzyme are different Therefore these inhibitors increase Km and decrease Vmax because they interfere with substrate binding and hamper catalysis in the ES complex 0 o Noncompetitive inhibitors bind to the active site and reduces the activity but does not affect the binding of the substrate Therefore the extent of inhibition depends on the concentration of the substrate o Noncompetitive inhibitors have identical affinities for E and ES They do not change Km but decreases Vmax
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