Kyle’s Section BIBC100 – Structural [email protected] Summer 2005Problem Set #61. Draw out a typical Lineweaver-Burk plot. Label the X and Y axes. Show how the plot would change as one increases the concentration of an inhibitor in the solution.Ans: X-axis = 1 / [S] , Y-axis = 1/Vo. The x-intercept will equal -1/Km, while the y-intercept will equal 1/Vmax. The slope of the line will equal Km/Vmax. Adding an inhibitor to this solution will increase the slop of the line. 2. Where does the enzyme “Chymotrypsin” carry out its function? That is, where in your body does this enzyme help break down proteins? Where is this enzyme synthesized?Ans: Chymotrypsin cleaves proteins in the small intestine lumen. It aids in the digestion of food. Chymotrypsin is synthesized in the pancreas. 3. What is the scissile bond?Ans: The scissile bond is the peptide bond of the substrate protein that is cleaved.4. Describe the 4 main structural elements of the active site of serine proteases. For each of these elements, describe its function.Ans: Structural Element FunctionMain Chain Substrate Binding Binds polypeptide segment adjacent to the region that is cleaved. Protein-protein binding results from backbone H-bondsSpecificity Pocket This pocket recognizes side chains (R-group) adjacent to the scissile bond. Oxyanion Hole This stabilizes the transition state substrate.That is, it stabilizes the O- group of the tetrahedral intermediateCatalytic Triad Forms the tetrahedral intermediate. Hydrolyzes the peptide bond.5. What kind of interaction or bonding takes place in the tetrahedral intermediate stage of a serine protease catalytic protein cleavage?Ans: covalent interaction6. Again, what is the order of the amino acid side chains that make up the “catalytic triad”. Which of these side chains is responsible for nucleophilically attacking the carbonyl carbon of the peptide bond that is broken? What intermediate structure doesthis create?Ans: Asp – His – Ser , Serine is responsible for nucleophilic attack. This attack creates the tetrahedral intermediate. 7. Is the “oxyanion hole” in a serine protease like chymotrypsin electronegative or electropositive? Delta-negative or Delta-positive dipole moments?Ans: electropositive, or delta-positive dipole moments.8. Chemically, which components of the chymotrypsin protease are responsible for this delta-positive dipole moments?Ans: the backbone N-H groups of Ser 195 (catalytic triad) and Gly 1939. Name three different types of serine proteases. For each, describe which amino acid(s) are found in its specificity pocket. Knowing this, which amino acid residues would each pocket best stabilize. Ans: chymotrypsin – Serine – bulky aromatic R groupsTrypsin – Aspartate - + charged R groupsElastase – Threonine and Valine – small, non-charged R groups10. What is the dissociation constant, Kd?Ans: This is a constant which describes how easily something will unbind from its acceptor. So, this could describe a ligand unbinding from a receptor, or a substrate unbinding from an enzyme without being catalytically altered. 11. In a ligand binding curve for oxygen binding, what is P50 equal to?Ans: P50 is the pressure of O2 (PO2) at which the enzyme is 50% bound to oxygen. 12. Define what Hemoglobin is and where it is found in your body. Define what Myoglobin is and where it is found in your body. Describe how these two proteins differ. Draw out what there respective oxygen dissociation curves would look like. Ans: Hemoglobin is an oxygen carrier protein found in your blood. Myoglobin is anoxygen binding protein found in red muscle tissue. Myoglobin has a much higher binding affinity than Hemoglobin. Hemoglobin is a tetramer, while myoglobin is a monomer 13. What is a “Heme” group? Draw out what it would typically look like.Ans: The heme group is the oxygen binding co-factor that is found in hemoglobin. Itis composed of a porphyrin ring (4 nitrogens) an iron center (Fe(II)), a histidine residue, and an oxygen binding site. 14. Describe the tetrameric structure of Hemoglobin.Ans: Hemoglobin is a pair of 2 alpha & beta dimmers. 15. What is the T-state of hemoglobin? What is the R-state?Ans: T stands for “tense” state. This is the state where O2 has not yet bound. The R-state is “relaxed” where at least one O2 unit has bound. R-state has a higher affinity for O2.16. What causes the dimmer-dimer rearrangement in the hemoglobin tetramer that is responsible for this change in affinity?Ans: F-helix shift upon oxygen binding. Subunits also rearrange contact
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