HIV Protease Enzyme System - Australia Assignments

Question 1: HIV Protease Enzyme System

Human immunodeficiency virus (HIV) was discovered in 1983 and instigates acquired immunodeficiency syndrome (AIDS). Found within the virus is HIV Protease that cleaves two 55 kDa and 160 kDa precursor polypeptides that are essential to the virus and involved in the viral life cycle. As a result of this, HIV Protease has been the target of therapeutics for many years.

Figure 1: Ribbon diagram of HIV Protease (1hvr.pdb)

Figure 1 is a ribbon structure representation of HIV Protease with the bonds of the catalytic residues Asp25 and Asp25’ (called Asp25-prime) shown as sticks in the centre.

An example polypeptide substrate for HIV-Protease is shown below in Figure 2 together with the active site side chain residues drawn below it. R and R’ corresponds to extensions of the polypeptide chain beyond the residues and bonds of interest.

Hydrolysis of the substrate peptide bond is initiated by a reaction with a single water molecule. The water molecule attacks the peptide bond because it is influenced by base catalysis provided by the side chain of Asp25’.

Figure 2: HIV-1 Protease Active Site and Substrate

(1) Parts of a catalysis mechanism for HIV-1 Protease is shown below. Complete the missing structures (points C and E) and curly arrows (points A, C, and E) in the mechanism.

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Question 2: Hen Egg White Lysozyme Enzyme System

Hen egg white (HEW) lysozyme is a small enzyme of 129 amino acids with a molecular weight of 14.5 kDa. It was the first enzyme structure to be solved (using x-ray crystallography in 1967).

Lysozyme acts as a mild anti-bacterial agent that works by breaking the glycosidic linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG): components found in bacterial cell walls. The structure of lysozyme is shown below.

Figure 3: HEW lysozyme structure, key residues A and B are shown in the active site cleft.

A pH dependence study of k_cat/K_M highlighted that the reaction rate was linked to the ionised state of two carboxyl side chain groups: one with a pK_a ~ 6 and one with a pK_a ~ 4. These amino acids are known to be at positions (A) and (B) in the figure above.

(2a) If lysozyme operates at pH 5, what is the predominant carboxyl side chain species (e.g. –COOH or –COO^–) for the pK_a ~ 6 side chain and the pK_a ~ 4 side chain?

The predominant species for the pK_a ~ 6 side chain at pH 5 is: ______________

The predominant species for the pK_a ~ 4 side chain at pH 5 is: ______________

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The pK_a values of all carboxyl side chain groups in HEW lysozyme were determined using 2D NMR spectroscopy and are listed below together with the secondary structure element they are found in:

Amino Acid	pK_a	Secondary structure element where amino acid resides
Asp 18	4.1	turn
Asp 48	6.2	turn
Asp 52	3.7	b
Asp 66	4.0	turn
Asp 87	4.1	turn
Asp 101	3.9	turn
Asp 119	4.0	a

Glu 7	4.2	a
Glu 35	6.2	a

(2b) As described earlier, key residues (A) and (B) in figure 3 are known to be either/or aspartic acid or glutamic acid. Using all the information you have been given, deduce the amino acids that are key residues (A) and (B).

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Key residues (A) in figure 3 is: ______________

Key residues (B) in figure 3 is: ______________

(2c) Parts of the mechanism for lysozyme are shown below. The catalytic lysozyme residue side chains can be identified as being above and below the polysaccharide chain shown at point A. Complete the missing structures (points B and E) and curly arrows (points A, B, C, D and E) in the mechanism, as well as from the knowledge you have gained, label the catalytic amino acids at point A in your mechanism with their correct name and residue number.

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Question 3: Analytical NMR

Look carefully at compound X below:

Figure 4: Compound X

(3a) Using letters (a, b, c, d etc.) label all of the carbons so that each unique carbon chemical environment on the structure above has a different letter.

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(3b) Sketch the 1D ¹H NMR spectrum you would expect for the compound X (including spin-spin couplings and other relevant information). On your spectrum, label each ¹H peak with the correct letter from part (3a) that defines the carbon to which each ¹H is attached.

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(3c) Sketch the 1D ¹³C NMR spectrum you would expect for compound X above (assume it is ¹H decoupled). On your spectrum, label each ¹³C peak with the correct letter from part (3a) that defines the carbon in the spectrum.

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(3d) Draw the DEPT-90 and DEPT-135 ¹³C spectra for compound X. Label each carbon peak using your notation from (3a).

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(3e) Sketch what you would think the ¹³C, ¹H HSQC spectrum of compound X would look like. Label each carbon peak using your notation from (3a).

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Question 4: Binding and protein NMR

Look at the ¹⁵N, ¹H HSQC spectra shown below. Both spectra were obtained two samples of the same 14.5 kDa protein at 25°C. SDS-PAGE of samples A and B show only pure protein that run at 14.5 kDa on the gel. Samples A and B samples were also analysed by protein sequencing and electrospray mass spectrometry and both techniques confirm the same protein present by weight and sequence.

(4a) What might have caused the differences in the NMR spectra in A and B?

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Compound Y is a potential drug candidate that is designed to inhibit the activity of protein in sample B above. In an attempt to determine the affinity of the binding interaction of compound Y to the protein in B, a series of ¹⁵N, ¹H HSQC spectra were collected at a protein concentration of 0.1 mM, and compound Y concentrations up to 5 mM. Some of the peaks are seen to shift as function of compound Y concentration as show below:

The overall changes in the chemical shift of one of the peaks is shown in the table below:

Concentration of compound Y / mM	NMR chemical shift change / 10³ · ppm
0.0	0.0
0.2	6.1
0.4	14.7
0.6	18.6
0.8	23.8
1.0	23.5
1.2	28.0
1.4	30.2
1.6	28.5
1.8	32.0
2.0	36.2
2.2	34.3
2.4	34.4
2.6	36.7
2.8	36.0
3.0	38.1
3.2	37.6
3.4	38.4
3.6	39.9
3.8	42.1
4.0	40.7
4.2	43.5
4.4	36.5
4.6	40.0
4.8	41.7
5.0	39.9

(4b) Draw the experimental binding isotherm data in a labelled figure showing the NMR chemical shift change as function of the concentration of the compound Y added.

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(4c) Based on the above experimental results, what is the likely affinity (K_d) of compound Y binding to the protein in sample B? Show in the same graph (from 4c) how the binding curve will look like with your estimated K_d value, and calculate how much of the protein is saturated with compound Y at 5 mM addition of Y.

You can use the following points to guide you:

Using all experimental information available, guess a possible value of K_d as well as a possible scaling factor linking the chemical shift change with % saturation.
Plot how such a binding curve would look like in the same graph as the data from 4c and compare. The use of graphing software such as Excel will simplify this task.

iii. Refine your guess until the binding curve represent the data as closely as possible.

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(4d) What is the standard free energy of association (ΔG°) for the above binding reaction? Show how you have calculated the ΔG° value.

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(4e) Is compound Y a good drug candidate? Why or why not?

[2]

End of assessment