Undergraduates, do any FIVE problems; graduate students, do SIX. The problems are due Wednesday, 29 April 2009. Please, please, please - be neat! Cite any sources you use.

1. Each of the questions following addresses an issue of prochirality and the stereochemistry of enzymatic reactions.

• A key sequence in the synthesis of valine in bacteria has been examined using isotopic labeling as shown:

  

  Does the replacement of the C-3 hydroxyl occur with retention of configuration or inversion? The C-2 hydroxyl? Explain.

• A study of the oxidation of serine was carried out using the racemic mixture of A and B shown. (The star indicates the isotopic label shown in the structures below.)

  

  D-amino acid oxidase will oxidize only serine having the R configuration at C-2; glycolate oxidase will remove only the pro-R hydrogen of glycolic acid. Does the final product contain tritium? Explain.

• Oxidation of polycyclic aromatic hydrocarbons by the cytochrome P-450 enzyme system produces diols, like that shown for the oxidation of naphthalene. Which prochiral faces of C-1 and C-2 were oxidized in this instance?

  

• Pig liver esterase selectively hydrolyzes the pro-R ester function of symmetrical diesters. Draw a stereoprojection of the product in the reaction below:

  

  2. Sorry about this, but if I didn't ask you to play with the Michaelis-Menten equation at least once, the ACS would disbar me, or whatever. The M-M equation is frequently used in alternative forms to produce linear graphs from which it is easy to obtain both V_max and K_m. Two such forms, the Lineweaver-Burke (L-B), and the Eadie-Hofstee (E-H), are shown below, along with the original.

Using the L-B format, a plot of 1/V vs 1/[S] should give a straight line of slope K_m/V_max; the point at which this line intersects [S] = 0 should be 1/V_max. The E-H version plots V vs V/[S], for a straight line having slope = -K_m,an intercept at V/[S] = 0 of V_max, and an intercept at V = 0 of V_max/K_m.

Fire up Excel, and use the data below and either the L-B or E-H method to obtain V_max and K_m.

  3. Mandelate racemase, as shown, catalyzes the racemization of mandelate. Atrolactate is a competitive inhibitor of the enzyme.

The picture below shows atrolactate bound at the active site of the enzyme, which also contains an Mg⁺⁺.

The second picture shows the active site with R-mandelate complexed, and the active site residues displayed.

The color code: Lys164 = yellow; His297 = blue; Glu221, 247, 317 = violet; Asp195 = dark green. Lys166 is also at the active site but not displayed to avoid obscuring 164; it lies just in front and above 164.

Some observations:

• If His297 is replaced in a site-directed mutation by a Gln, the enzyme no longer catalyzes the racemization of the R-mandelate, but still racemizes the S-.

• Contrariwise, if the Lys166 is replaced by an Ala, the R- is still racemized, but the S- is not.

1. Write a mechanism for the non-enzymatic racemization in basic aqueous solution; can you suggest a reason why this racemization is quite slow?

2. Why does atrolactate inhibit the enzyme?

3. Explain the results of the site-directed mutations.

References that may be useful: Landro, Biochemistry, 1991, 30, 9274, 1994, 33, 14213; Whitman, Biochemistry, 1985, 24, 3936.

  4. Lysozyme is a widely distributed enzyme (tears, saliva) that acts as an antibacterial agent by hydrolyzing the polysaccharides of bacterial cell walls:

Note the retention of configuration at the reaction site, and the structure of the transition state inhibitor..

The active site, shown below with a substrate bound, consists of Glu35 and Asp52; initially, Asp52 is ionized, whereas Glu35 is not.

Galactosidase catalyzes the same reaction of polysaccharides lacking the N-acetyl group, and uses the same functional residues to do so. However, in this case there is evidence that the intermediate is covalent. Keeping in mind that two inversions accomplish a retention:

• Write the mechanism for lysozyme activity; and

• Decide how to modify it for galactosidase. Explain!

  5. The monoamine oxidases are enzymes responsible for the deamination of monoamines such as adrenaline and dopamine, that act as neurotransmitters. These enzymes require FAD as a cofactor. Parkinson's disease is associated with lowered levels of dopamine, and clinical depression may be caused by lower levels of other amines. Hence, an inhibitor of these enzymes would have some clinical value.

Deprenyl, below, is a suicide ("Trojan Horse") inhibitor of monoamine oxidases. Consult Maycock, Biochemistry, 1976, 15, 114, and suggest a mechanism for this inhibition. Another useful source of help would be to download the structure of human monoamine oxidase from the PDB, structure 1gos, and read the paper reporting the structure.

  6. We have looked at several metalloenzymes this semester, but paid little attention to how the metal is bound to the enzyme. Superoxide dismutase (pdb 1b4l) contains a copper ion coordinated to the side chains of three nearby residues; fungal laccases (pdb 1gw0) contain two copper sites, one with three coppers and one having only one. Use Rasmol to visualize either one of these enzymes, and determine what amino acids supply the ligands for the metal ions. Then prepare a nice graphic to hand in, showing the protein and the ligands that coordinate the metal ion.

  7. (a) In the active site of chymotrypsin, one oxygen of the Asp102 carboxylate is hydrogen bonded to the ring NH of imidazole, as we have seen. The other oxygen of the carboxylate is hydrogen bonded to the backbone NHs of Ala55 and Ala56. What would be the effect on the enzyme of mutating Asp102 to Asn?

(b) We showed in class that the catalytic activity of subtilisin is much reduced by mutations of any or all of the members of the "catalytic triad". In particular, mutation of Ser221 to Ala reduced the activity by about a million-fold - but NOT to zero. Can you suggest a mechanism that would still allow this mutant to hydrolyze a peptide?

8. The SuMo web site at IBCP in France accepts an input protein structure in pdb format and searches the Protein DataBank for other structures with similar ligand binding sites. The accession number of a pdb file can be entered, or you can upload a file from your own computer. The software will search the protein for binding sites and offer you the chance to select those you wish to use. You can wait for a result on the web, and you will be emailed the URL of a web page with the results if you don't wish to wait.

Submit the structure of calpain (1kxr) with four calciums bound. Select the four Ca binding sites as the basis of your search. You will get several hundred hits. Select the best three of your hits. Download the pdb files for your hits. Make Rasmol views showing the Ca coordination lignads. Submit the resulting pictures with your results.

9. Determine the sequences of the activation segments of carboxypeptidases A and B, and thermolysin. Then do a sequence alignment. What residues are conserved, if any? Submit the sequences to PsiPred or NNPredict for prediction of their secondary structures. What similarities are predicted, if any?

10. Chymotrypsin is used to hydrolyze the dipeptide Phe-Ala. The carbonyl oxygen of the Phe is labeled with ¹⁷O. Where will the label be found in the products? If the same dipeptide is hydrolyzed in aqueous acid, where will the label wind up? Write as much of a mechanism for each reaction as may be necessary to justify your answers.

11. Structures are available in the Protein Databank for HMGCoA reductase complexed with the statins Compactin, Simvastatin, and Fluvastatin.

(a) Compare the structures of the enzyme with and without the inhibitors bound, and identify any significant differences.

(b) Identify the residues that seem to coordinate the statins. Are any of these catalytic residues? Are any of them involved in binding the NADP(H)?

12. We discussed the mechanisms of action of both prokaryotic squalene cyclase and human oxidosqualene cyclase. Download the structures of these proteins from the PDB.

(a) Characterize the overall structures in terms of domains, folds, and so on. The SCOP website will help.

(b) We found about 25% sequence identity between the two enzymes. Identify the conserved residues that are part of the catalytic site and the binding site. Create high-quality graphics that show those conserved residues.

(Papers that may help: Chem. Comm., 2002, 291; Science, 1997, 277, 1811)

13. Divergent evolution, as we described for the serine proteases, can lead to enzymes with different selectivities for the same reaction type. It also can lead to enzymes of closely similar structure that catalyze different reactions!

Read Ann. Rev. Biochem., 2001, 70, 209, and describe the sequences, structures, and reactions of a pair of enzymes that fit this latter description. That is, provide sequence alignments, structure alignments, and descriptions of the reactions catalyzed by the pair.

14. The protein avidin,, found in egg whites, binds the essential human nutrient biotin so tightly that excess egg consumption can lead to biotin deficiency.

• What is the structure of biotin, and what is its function?

• Examine a crystal structure of avidin complexed to biotin and identify the mode of binding, including the specific residues involved

• Enzymes that employ biotin as a cofactor can be isolated easily by affinity chromatography involving avidin. Explain how this would work