MCB354FA23_PS02

1 MCB 354 Name: _____________________________ Fall 2023 Discussion Problem Set 02 Question 1 Activity of the bacterial enzyme acetoacetate decarboxylase sharply declines below pH 6, suggesting there is an ionizable catalytic residue with pKa ~ 6. Using mutagenesis, this critical catalytic residue was determined to be Lys-115. a. What is the typical pKa of a lysine side chain and what ratio of deprotonated to protonated lysine would be expected at pH 7.5 (standard pH inside a bacterium)? b. For catalytic Lys-115, what is the ratio of deprotonated to protonated side chain at pH 7.5? c. The neighboring residue at position 116 is also lysine. Explain how this perturbs the pKa of the catalytic lysine residue. d. Lys-116 is mutated to glutamate. How is the pKa of catalytic Lys-115 expected to change and why? HA , H + A ka = CH + ] [A] - > logka = log(P SHA] - 109 , /H + ) + logir(2) = 10910 [H + = -logiokatlog() = PH = pka + 10910( CA-] : concentration of Conj base ⊥ typical pka of a lysine side chain : 10 5 (pH = pka + 109/C)]ciation CHA] : concentration of acid it 25 7 . 570 S + log (ly3 NH2)] in acteria deprotonated protonated ⊥ ratio : 1017 . 5-10 . ) = 0 . 001 ↑ very small amount of lysine deprotonates inside bacteria PH = pKa + log (3) A] Catalytic : 7 . 5-6 Hog (i NH = 1 . 4 ⊥ ratio : 1047 . 5-6) 30 a lot of Catalytic lysine deprotonates ⊥ if there are 2 positive neighboring residues then the ionization state would become destabilized due to the electrostatic repulsion resulting in a shift of the equilibrium . In doing so , the pka decreates , resulting in a stronger acid Chemical environment of lysines would repel each other positive : 4 acidic ⊥ Considering glutamate is negatively charged , the protonated form would be stabilized unlike when the 2 neighboring residues negative : ↑ basic Were both of the same charge . As a result of this change , the pla will increase , resulting in a stronger base attraction reaction - > stabilizes the base

2 Question 2 Src is an important signaling protein that is regulated by phosphorylation. A purified solution of Src is digested with proteases into peptides, and the peptide mixture is then incubated with extracts from lymphoma cells known to have tyrosine kinase activity. The peptides are analyzed by mass spec to identify any Src peptides that are phosphorylated. Src peptide fragment Asn-Gln-Tyr-Thr is identified. a. Draw basic stick figures showing the ionization states of peptide Asn-Gln-Tyr- Thr as the pH is raised from acidic to basic. Indicate the pKa values of the different ionizable groups on your figure.

4 Question 3 Peptidyl prolyl isomerase (PPI) catalyzes the interconversion between cis and trans proline isomers. This can be a rate-limiting step in the folding of some proteins. a. Draw resonance structures of the amide bond (with arrows for electron pushing) and explain why there is restricted rotation. b. Draw a proline residue in the cis isomer.

5 Question 4 Proliferating Cell Nuclear Antigen (PCNA) is a ring-shaped protein that forms a clamp around DNA. DNA polymerase binds to the outside of the PCNA clamp, keeping the polymerase tightly associated with DNA during replication. PCNA is a homo-oligomer; each subunit is shown as a different color in the image below. a. In the lab, you purify PCNA from mouse tissue. Based on the protein sequence, the extinction coefficient for a single PCNA molecule at 280 nm is e = 15,930 M -1 cm -1 . What is the extinction coefficient for the homo-oligomeric PCNA clamp? b. Your purified sample of murine PCNA absorbs 72% of incident light at 280 nm using a standard 1 cm path length cuvette. What is the concentration in micromolar?

7 d. The gliadin peptides used in diagnostic tests are partially deamidated; these modified peptides better recognize antibodies in the patient’s blood. Deamidation is the conversion of a primary amide functional group to a carboxylic acid. Which amino acid in the gliadin peptide can be deamidated and what amino acid will it become? Draw both of these amino acids in solution at pH 7, showing correct stereochemistry around the alpha carbon. e. Compared to the original sequence, will the isoelectric point of a deamidated peptide decrease, increase, or stay the same?

8 Additional Practice Question 6 Substance P binds its receptor, the neurokinin 1 receptor (NK1R), with a dissociation constant K D = 0.5 nM. Based on a NMR structure, a serine residue of NK1R forms a hydrogen bond with Substance P. a. To which amino acid would you mutate the serine to test the importance of the hydrogen bond and why? b. If the K D of mutant NK1R binding to substance P increases to 25 nM, what was the energy contribution of the hydrogen bond? (R = 8.3 J mol -1 K -1 and assume T = 310 K / 37 ° C)

10 b. What is the isoelectric point of gluten exorphin B4? c. Gluten exorphins can be detected in the urine (pH ~ 6) of autistic children. Draw the structure of gluten exorphin B4 at pH 6.0. Show correct stereochemistry around the alpha carbon. d. Gluten exorphin B4 binds the opiate receptor in the brain with K D = 4.1 μ M at physiological temperature (37 ° C). What is the change in free energy for the binding reaction?

11 Additional Practice Question 8 Leucine enkephalin is a peptide neurotransmitter in the brain that stimulates opioid receptors, controlling mood and feelings of pain. It has the amino acid sequence YGGFL. a. What are the ionizable groups on leu-enkephalin, and what are their p K a values? b. Draw a basic diagram showing the titration of leu-enkephalin from low pH to high pH with NaOH. Clearly indicate which groups are changing ionization state and the net charge of the peptide as the pH increases. c. What is the isoelectric point of leu-enkephalin?