You are advised to spend an equal amount of time on each question.

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1 UNIVERSITY OF EAST ANGLIA School of Chemistry Main Series UG Examination BIOPHYSICAL CHEMISTRY CHE-5601Y Time allowed: 2 hours Answer THREE questions. You are advised to spend an equal amount of time on each question. All questions carry an equal number of marks. Answer EACH question in a SEPARATE answer book. The breakdown of marks within each question is indicated by the percentage figures in brackets on the right. The following are provided: Graph Paper Physical Constants on page 2 of this booklet. Do not remove this question paper from the examinations room. Notes are not permitted in this examination. Do not turn over until you are told to do so by the Invigilator. Module co-ordinator: Nick LeBrun (CHE) Copyright of the University of East Anglia Version 1

2 2 Physical Constants Speed of light, c0 = m s 1 Gas constant, R = J K 1 mol 1 Faraday s constant, F = C mol 1 Avogadro s constant, NA = mol 1 Planck s constant, h = J s Boltzmann s constant, k = J K 1 Elementary charge, e = C Permittivity of free space, ε0 = J 1 C 2 m 1 Density of water at 25 C, ρ = g ml 1 Version 1

3 3 1. Answer BOTH parts. (a) In the presence of the enzyme lactate dehydrogenase, pyruvate is reduced by nicotinamide adenine dinucleotide (NAD) according to the equation: Pyruvate + NADH + H + Lactate + NAD + G for this reaction is 25.1 kj mol -1 at 298 K. (i) Define the meaning of G and explain how it differs from G. [20%] (ii) What is the significance of the negative sign for the value of G in the reaction above? [10%] (iii) Calculate the value of the equilibrium constant, K, for the reaction at ph 7 and 298 K. [20%] (iv) The concentrations of the reaction components at ph 7 are as indicated in the table below. Calculate the value of G for the reaction under these conditions. Reaction component Concentration (M) Pyruvate NADH Lactate NAD [20%] (b) Cu 2+ was found to bind reversibly to a protein, P, and the following data were obtained: Free [Cu 2+ ](µm) [Cu 2+ -P](µM) The total concentration of P ([P]total) was M throughout the titration. Use the Scatchard equation below to graphically determine the value of Kd for binding of Cu 2+ to P. r [Cu 2+ = ] 1 K d r K d [30%] TURN OVER Version 1

4 4 2. Answer BOTH parts. (a) Many reactions involving enzymes are reversible processes in which the enzyme catalyses the interconversion of substrate and product as summarised below. Enzyme + Substrate Enzyme:Substrate Enzyme + Product Outline the key features of the experimental conditions typically employed to simplify the analysis of kinetic data acquired for such systems. [15%] (b) The following data were obtained for an enzyme catalysed reaction: [Substrate] (µm) Initial rate, ᴠ (µm s 1 ) Using the Hanes-Woolf equation below, graphically determine the parameters Vmax and KM. [S] [S] = ν V max K V M max [40%] (c) Molecule X was used as a substrate analogue in an attempt to structurally characterise the Enzyme:Substrate complex. X was found to inhibit the enzyme as detailed below: (i) State the mechanism by which X inhibits the enzyme. Explain your reasoning. [15%] question 2 continues / Version 1

5 5 /question 2 continued (ii) Calculate the dissociation constant, KdI, describing the binding of X to the enzyme. [15%] (iii) Comment on whether X is a sensible choice of molecule to mimic the binding of substrate to the enzyme. [15%] 3. Answer BOTH parts. (a) Copper and nicotinamide adenine dinucleotide (NAD) are redox active in nature. The biochemical standard reduction potential for the Cu 2+ /Cu + couple is E = V, while that for the NAD + /NADH couple is E = 0.32 V. The reduction of Cu 2+ by one molecule of NADH can be written as: NADH + 2Cu 2+ NAD + + 2Cu + + H + (i) Which of the four species (NAD +, NADH, Cu 2+, Cu + ) is the strongest reductant, and why? [10%] (ii) Write half-reactions for the NAD + /NADH and Cu 2+ /Cu + couples. [20%] (iii) Calculate the cell potential, E, for the reduction of Cu 2+ by NADH. [15%] (iv) Calculate the value of G for the reduction of Cu 2+ by NADH. [20%] (b) Cytoglobin is one of the most recently discovered members of the globin family of proteins. In addition to a single heme group, it contains an internal disulfide bond that may be important for function. The molecular mass of the protein was determined to be approximately 21,400 Da. (i) Provide a brief description of a technique other than mass spectrometry that could have been used to determine the approximate mass of cytoglobin. Explain how you would perform such a mass determination. [20%] (ii) The mass of the protein was more accurately determined using electrospray ionisation mass spectrometry (ESI-MS) as 21,404.7 (±0.3) Da after treatment with dithiothreitol (DTT). Upon removal of DTT, the protein mass was observed to be 21,402.6 (±0.3) Da. Provide an explanation for this observation. [15%] TURN OVER Version 1

6 6 4. Answer ALL parts (a) to (c). (a) Electron paramagnetic resonance (EPR) spectroscopy can be used to study molecules that contain an unpaired electron spin. (i) Draw an energy level diagram to represent the EPR experiment for an S = ½ system and describe how a spectrum is recorded. [25%] (ii) Using the equation: E = gβb calculate the g-value of an EPR feature that is centred at a magnetic field of 250 mt in a spectrum recorded using microwaves of frequency ν = s 1. Note that the Bohr magneton, β = J T 1. [10%] (iii) The molecule below is MTSL. This carries an unpaired electron and can be attached to cysteine residues in order to spin label the protein. N O O S S O Sketch and explain the form of the EPR spectrum of MTSL. [15%] (b) The 1 H NMR spectrum of cytochrome c shows good spectral dispersion, with peaks below 0 ppm, and peaks in the amide region up to 11 ppm. In the presence of 4 M guanidinium hydrochloride, however, the spectrum shows no peaks below 0 ppm, and poor spectral dispersion in the amide region, with no peaks above 8.5 ppm. Explain these NMR spectra in terms of the cytochrome c structure. [15%] question 4 continues / Version 1

7 7 /question 4 continued (c) To further study the structure of cytochrome c by NMR spectroscopy, isotopic labelling is required. (i) What is isotopic labelling and how is it achieved for protein NMR samples? [10%] (ii) Why is it necessary to use isotopic labelling to study protein structures by NMR spectroscopy? [10%] (iii) You have obtained a sample of isotopically labelled protein with a known sequence of amino acids for structural studies by NMR spectroscopy. Briefly describe the remaining steps required to solve the conformational structure of this protein. [15%] END OF PAPER Version 1

You are advised to spend an equal amount of time on each question. All questions carry an equal number of marks.

UNIVERSITY OF EAST ANGLIA School of Chemistry Main Series UG Examination 2014-15 INORGANIC CHEMISTRY CHE-2C32/5301B Time allowed: 2 hours Answer THREE questions. You are advised to spend an equal amount