CChheemm 44334433 FFiinnaall Exam 2009 Name : Answer any seven of questions 1-8 . Each question is worth 15 points for a total of 105 points. 1. (i) For the HIV protease enzyme, the natural substrate and two drugs are shown on the far right, along with the Val residue in the binding pocket. Given the kinetic data on the right identify, giving your reasoning, which line belongs to which drug and calculate KM for the reaction with normal substrate. (ii) For the enzymatic reaction on the right draw the mechanism and suggest specific amino acids for A and B in the diagram above . (iii) Lysozyme is a small enzyme which hydrolyzes polysaccharide chains using amino acids Glu35 and Asp52 as shown below. Identify, giving your reasoning the optimum pH range for this reaction and comment on the nature of the ES complex that is formed2. (i) Identify, giving your reasoning, an appropriate pH range for the enzymatic reaction shown on the right and comment on the role of the Zn2+ present in the active site. (ii) Both enzymes shown to the right initiate catalysis by attack in the substrate. Show the initial mechanism for both the Serine Protease and the HIV Protease and identify any differences between the optimum pH for each process. (iii) In the hydrolysis reaction shown below the same substrate is shown bound to three different enzymes, the normal or wild type enzyme (wt) and two mutants that have one amino acid changed in the active site. Determine, giving your reasoning, which column of kinetic data belongs to which mutant and calculate KM and Vmax for the wild type enzymatic assay [S] V(wt) V V (mM) ([S]/s) ([S]/s) ([S]/s) 0.5 33 11 17 1 50 20 25 2 67 33 33 10 91 71 45 20 95 83 48 50 98 93 49 75 99 95 493. (i) Line up one of the RNA nucleotides below in the box on the right in such a way as to give the best h-bonded interaction between the two bases. (ii) For the DNA micrograph image on the right what level of structure does this represent and what function(s) does the above structure serve in the organism (iii) The positively charged histones, shown on the right, are used to stabilize DNA. Explain in terms of forces and explain how acetylation of these histones , also shown, will affect the TM of the DNA 4. (i) A mutation was made in the protein, such that a completely buried Trptophan residue was replaced by an Alanine residue. This change did not affect the overall structure of the protein but the measured ∆H for folding to the native structure increased by 5 kJ/mol in the mutant protein. Give a possible reason for this increase in enthalphy and explain any other possible effects on ∆G for folding the protein into its native structure.(ii) Calculate, showing your method, the isoelectric point for the peptide shown on the right : (iii) Succinic acid, shown on the right, is a diacid and with a 1st pKa = 5.0 and a 2nd pKA = 7.0. Explain why the second pKa is so much higher than the first and calculate, showing your method, the pH at which 90% of the molecule has a charge of -1. 5. (i) For the following peptide sequence : Pro-Phe-Glu-Ala-Ala-Met-Cys-Lys-Trp-Tyr-Ala-Arg-Pro-Asp-Gly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Identify, giving your reasoning, a small (<8 residue) α-helix and a turn. (ii) The structures for the amino acid tryptophan, I, and the closely related compound indole, II, are given below. Explain the observation that at pH=7 tryptophan crosses a cell membrane 1000 times more slowly than indole : (iii) For the oxygen binding curve on the right identify what the axis “Y” is measuring and draw the appropriate binding curves for: (a) Fetal Hb (b) Hb with added biphoshpho glycerate (also called diphosphoglycerate) (c) Myoglobin I2HN COOHHCCH2NHIIHN(iv) Identify the conditions necessary to go from the protein structure on the left all the way to the structure on the right 6. (i) The protein on the right contains a Thr at position A. Predict in terms of forces the effect on ∆G for folding to the native structure, when this Thr is replaced by Val (ii) The C-terminus residues Tyr145-His146 of the β-chain of Hb sits in the cavity shown on the right, Draw this 2 amino acid fragment in such a way so as to maximize the interactions with the Lys, Asp and Val shown (iii) The melting curves for two proteins, one with Thr at position 120 (square) and one with Cys at 120 (diamond) are shown on the right. Propose one reason for the difference in TM and (v) The antibody shown below binds specifically to the antigens shown NAG. Within the diagram itself, on the right, identify all the forces responsible for Ab:Ag bindingLys40Asp94CVal98ONH3+COO-AFor Questions 7-8 indicate all answers (i) through (v) that are correct. 7. Which, if any, of the following statements are true for binding plot of myoglobin (Myo), normal hemoglobin (Hb-normal) and a mutant hemoglobin (Hb-mutant) shown below: (i) The mutant hemoglobin favors the R state (ii) The myoglobin shown preferentially adopts the Tense, or T, state (iii) The mutant hemoglobin binds less oxygen in the lungs but releases oxygen more efficiently in the tissues (iv) The normal hemoglobin binds more oxygen in the lungs but releases oxygen less efficiently in the tissues (v) A loss of any of the positively charged amino acids in the central cavity of Hb (page 229 of your text) could have produced the mutant Hb whose plot is shown above Comment: 8. Which of the following statements is true for the enzymatic reaction : (i) The reaction velocity for formation of products is directly proportional to kcat (ii) The reaction velocity for formation of products is directly proportional to Km (iii) The maximum reaction velocity for formation of products is independent of substrate concentration (iv) The maximum reaction velocity for formation of products is independent of enzyme concentration (v) Competitive inhibitors change the observed kcat but not the observed Km Comment E + Pkcatk-1k1ESE +