Exam 2 SG: Enzymes, Serine Proteases, Reaction Rates, Inhibition,

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University of Massachusetts Amherst
Biochemistry & Molecular Bio.
David Gross

BIOCHEM 523 EXAM 2 STUDY GUIDE FALL 2013 When and Where: The second exam in Biochem 523 this fall will be held on Monday, October 21 from 7:00-9:00 p.m. in Room 20, Hasbrouck. Although we have allocated 2 hours for the exam, we expect that most of you will be able to complete it more quickly. Material Covered: The exam will cover all material discussed in class from Chapter 5 through Chapter 7 in Pratt & Cornely's Essential Biochemistry and the articles on Xeroderma pigmentosum. Time Conflicts: In the event of a conflict that makes it impossible for you to take the exam at 7:00 p.m. please inform me right away so that I can make alternative arrangements. In general, makeup exams will take place before the scheduled exam, either in the morning or in the afternoon. EOC Questions: Some end-of-chapter questions for study and practice. There is a wide variety and not all are of then same quality, though they’re useful for review or a challenge. Chapter 5: 3,7,9,12,13,17,19,25,35,42,46,64,71(Review); 39,49,53,73,74 (Challenge) Chapter 6: 1,6,10,11,19,21,24,26,29,34,46,57,61,67 (Review); 17,25,39,40,52 (Challenge) Chapter 7: 3,4,9,10,12,13,14,22,25,26,27,28,31,33,41,44,55 (Review); 11,18,37,50,54,59 (Challenge) Structures: I trust you will not have forgotten what group (hydrophobic, polar, charged) each of the 20 amino acids belongs to, nor how to write the structures for at least one amino acid from each group, specifically valine (hydrophobic), glutamine (polar) and argiunine (charged), as well as several amino acids with special structural/functional roles: methionine, histidine, proline, cysteine and tyrosine. In addition, I would like you to know the pKa’sfor the N-terminal amino group, for the C-terminal carboxyl group, and for the side chains of aspartic acid, histidine and lysine. Regrade Policy. Our policy on exam regrades is as follows: 1. Regrade requests will be accepted only when a grading error has been made. 2. All requests for exam regrades must be made by 12:30 p.m., one week after the exam is returned. 3. Your request should be typed, indicate which question is in dispute, and what is in dispute. Arithmetic errors are easily corrected. Argumentative requests will not get very far; we always try to grade your exams as leniently as possible. 4. Give your request, along with your exam, to Prof. Zimmermann before or after class. Alternatively, ask someone in the BMB Department office (9th floor LGRT) to put it in his mailbox. THINGS TO KNOW FOR EXAM 2 Chapter 5 •Myoglobin (single subunit, muscle) Heme: nature of coordination bonds between Fe and porphyrin nitrogens • Has 6 possible coordination bonds • 4 N atoms of porphyrin ring coordinate bond with heme Oxygen binding: fractional saturation (Y) and oxygen partial pressure (2O ) • The proportion of total myoglobin molecules that have bound O2 is the fractional saturation (Y) • Y=[MbO2]/([Mb]+[MbO2]) (Mb = myoglobin) • pO2 is the partial pressure of oxygen (torr) • Y=pO2/(K+pO2) (K=([Mb][O2])/[MbO2]) • The amount of O2 bound to myoglobin is a function of the oxygen concentration and the affinity of myogloving for O2 Oxygen binding curve: hyperbolic • As O2 concentration increases more and more O2 molecules bind to the heme groups until at very high O2 concentrations virtually all heme groups have been bound • Changes in Mb structure upon oxygen binding • AHis residue on the E7 alpha-helix forms a H-bond w/ O2 when it binds •Hemoglobin (heterotetramer with two different chains; red blood cells) Evolutionary relationship of Hb and Mb • Looks a lot like myoglobin • Hemoglobin alpha- & beta-chains and myoglobin have similar tertiary structures • All have heme group in hydrophobic pocket, a His F8 that ligands the Fe(II) ion, and a His E7 that forms a H-bond with O2 • TheA.A. sequence is only 18% similar between the 3 however • Invariant residues are those that are identical in all the glovins and are essential for the structure and function (can’t be replaced) • Conservatively substituted areas are under less selective pressure andA.A.’s can be switched out by a similarA.A. • Other positions are viriable and can accommodate a variety of residues Oxygen binding • Binding is cooperative, binding of one O2 molecule increases the affinity for another O2 molecule • @ low O2 concentrations hemoglobin is reluctant to bind the first O2 molecule • Hemoglobin’s four heme groups are not independent but communicate with each other in order to work in a unified fashion Oxygen binding curve: sigmoidal • @ low O2 concentrations the affinity of hemoglobin for O2 is low, as O2 concentration increases the saturation of hemoglobin increases sharply indicating cooperative binding, until virtually all hemoglobin molecules have been bound by O2 • Cooperative binding of oxygens to the four subunits • The 4 O2 taken up by hemoglobin binds with about 100X greater affinity than the first • In lungs where pO2 is high, hemoglobin is very highly saturated, the pO2 in tissue is less, hemoglobin is not highly bound to O2, then myoglobin takes up O2 and distributes it • In deoxyhemoglobin the heme Fe ion has 5 ligands, the porphyrin ring is dome-shaped, Fe lies bowed slightly toward His F8 • Oxyhemoglobin has 6 ligands, moves into the center of the porphyrin plane, pulls His F8 toward the heme group, drags entire F helix • The entire protein must move for this to happen, results in rotation of one alpha-beta unit relative to the other • Hemoglobin has 2 quaternary structures, deoxyhemoglobin is “tense” (T), oxyhemoglobin is “relaxed” (R) • Whenn one molecule binds, subsequent molecules bind easier because hemoglobin is already in the R conformation Regulation of oxygen binding Bohr Effect • Normally when O2 binds to hemoglobin H+ is released, decreasing pH • Therefore increasing the pH of a solution of hemoglobin favors O2 binding and pushes the reaction to the right • Decreasing the pH favors O2 dissociation and pushes the reaction to the left • The reduction of hemoglobin’s oxygen-binding affinity when the pH decreases is known as the Bohr effect Role of 2,3,-bisphoisphoglycerate • Binds in central cavity of hemoglobin, only in T state • 5 negative charges on BPG interact w/ positively charged groups in deoxyhemoglobin • The presence of BPG stabilizes the deoxy conformation • W/o BPG hemoglobin would bind O2 too tightly to release it to cells Fetal hemobglobin differs slightly from adult hemoglobin in structure; higher O2affinity • has the composition alpha2,gamma2 • One of the His residues that binds to BPG not pressent in gamma strand, decreased BPG binding • Hemoglobin has a higher O2 affinity than adult hemoglobin for O2, helps transfer O2 from maternal circulation across placenta to fetus •Nature and importance of cytoskeleton • Formed from microfilaments (actin), microtubules (tubulin), and intermediate filaments (collagen) • Collagen fibers provide extracellular support in multicellular organisms • Microfilaments and microtubules also play a role in motion of cell •Pattern of assembly of actin into microfilaments • Globular protein • Polymerized actin called F-actin, monomeric globular form called G-actin • Each actin subunit has same orientation,fibers have distince polarity, end w/ ATP site = - end, opposite end = + end • subunits polymerize in a right handed helix role ofATP in polymerization of actin monomers • most of actin subunits in a microfilament contain boundADP b/c the polymerization rxn is catalyzed by F-actin but not by G-actin • only most recently added subunits still containATP • some proteins may be able to distinguish rapidly polymerizing (ATP rich) microfilaments from longer established (ADP rich) ones •Pattern of assembly of - and -tubulin into microtubules • First tubulin dimers associate for form short linear protofilaments • Protofilaments allign side to side in a curved sheet, which wraps around on itself to form a hollow tube of 13 protofilaments • role of GTP in polymerization of -+-tubulin dimers • each tubulin subunit contains a nucleotide binding site for either GTP or GDP • When dimers form, alpha-subunit binding site becomes burried • After dimer incorporated into a microtubule, beta-binding site also sequestered by dimer on top of it • GTP is then hydrolyzed, but resulting GDP remains bound to the tubulin b/c can’t diffuse away • alpha-subunit GTP is not hydrolyzed and remains where it is •Pattern of assembly of keratin into intermediate filaments • Each subunit contains an alpha-helix flanked by nonhelical regions @ N- & C- termini • Two polypeptides interact parallel to form coiled coil • Dimers then associate in staggered antiparallel arrangement and form higher-order fivrous structures • No nucleotides required for intermediate filament assembly, the N- & C-terminal domains help align the subunits • The fibers are crosslinked by disulfide bonds btwn Cys residues structural unit is dimer of  helices in a parallel coiled coil with left-handed twist • A.A. sequence is 7-residue repeating subunit w/ 1 & 4 subunits nonpolar • These nonpolar residues line up along one side, appear every 3.5 residues • Since there are 3.6 residues per turn, strip of nonpolar resudues winds around surface of helix • Two helices w/ nonpolar strips in contact for a left handed helix •Nature and importance of collagen • Plays a major structural role in extracellular matrix, in connective tissue within and between organs, and in bone. primary structure: Gly-Pro-Hyp repeat • Gly only has H as a side chain, can normally adopt wide range of secondary structures • The imino groups of Pro and Hyp residues constrain the geometry of the peptide formation of right-handed, parallel triple helix • Gly residues all at the center of the tripls helix role of sequence repeat in forming the triple helix • Chains are parallel but staggered by 1 residue so that Gly appears @ every position along the axis of the triple helix pattern of H-bonds in triple helix • The backbone N-H group of each Gly residue is linked to the backbone C=O group in another chain • The other backbone N-H and C=O groups of the same residues are unable to connect but link w/ a network of water molecules surrounding the triple helix stabilization of triple helix by chemical cross-links • The cross-links are covalent bonds between side chains that have been chemically modified following polypeptide synthesis •Myosin (molecular motor) anatomy of myosin heavy chain dimer: head, neck and tail (coiled coil) • role of myosin light chain in reinforcing neck • The light chains stiffen the neck so that it can act as a lever the reinforced neck as lever arm movement of myosin relative to actin thin filaments in muscle 'contraction' • Each head can interact noncovalently w/ a subunit in an actin filament, but 2 heads act independently, only 1 head binds at a time • The movement of the myosin is towards the + end of the actin filament role ofATP in muscle 'contraction' ("hopping") • ATP hydrolysis •Kinesin (molecular motor) anatomy of kinesin heavy chain dimer: head, nck and tail (coiled coil) functions with kinesin light chains to transport cellular "cargo" on microtubule "tracks" role ofATP in vesicle transport ("walking") Chapter 6 •Ways to increase chemical reaction rates • Increasing the temperature (adding energy in the form of heat) • Increasing the concentrations of the reacting substances • Adding a catalyst (a substance that participates in the reaction yet emerges at the end in its original form) •Nature of enzymes as catalysts • most enzymes are proteins, but a few are RNA’s (ribozymes) achieve spectacular rate enhancement • chymotrypsin as an example catalyzes the hydrolysis of peptide bonds @ a rate 11 about 1.7∗10 times faster than normal • rate enhancements of 10 −10 12 are typical high substrate specificity • the functional groups in the active site of enzymes are carefully arranged so that the enzyme can distinguish its substrate from among many others and can mediate a single chemical reaction amenable to numerous kinds of regulation • they are regulated so that the organism can respond to changing conditions or follow genetically determined developmental programs •Simple model reactions, often colorimetric, used to identify, quantify enzymes •Nomenclature and classification • enzymes are formally classified into 6 subgroups o oxidoreductases: oxidation-reduction reactions o transferases: transfer of functional groups o hydrolases: hydrolysis reactions o lyases: group elimination to form double bonds o isomerases: isomerization reactions o ligases: bond formation coupled with ATP hydrolysis • some enzymes are named by adding suffix –ase into name of its substrate •Reaction coordinate diagrams meaning of free energy of activation, ∆G‡ • the energy-requiring step of the reaction (the energy barrier) • reaction needs to overcome this to reach the transition state significance of transition state • this is the point of highest energy, an intermediate between reactants and products • lifetimes are extremely short because of their instability relationship between ∆G‡ and reaction rate • the higher the activation energy barrier, the less likely a reaction is to occur (the slower it is) •How enzymes reduce the activation energy barrier • a catalyst decreases the activation energy barrier by interacting w/ the reacting molecules such that they are more likely to assume the transition state • an enzyme doesn’t alter the net free energy change of a reaction, it provides a pathway from reactants to products that passes through a transition state of lower free energy than the uncatalyzed reaction • an enzyme lowers the height of the activation energy barrier by lowering the energy of the transit
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