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:

  

Remember that changes in substituent priority can change the configuration designator without changing absolute configuration.

2. 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.

3. 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.

  4. 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 carboxypeptidase B (1zg8) or thermolysin (1tlx). Select the Zn as the basis of your search. You will get several dozen hits. Select the best three of your hits and download their PDB files. Use Jmol to make views showing the ligands coordinating the Zn, and describe the functions of the proteins. How similar aare the sequences? How similar are the overall structures?

5. 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.

6. The first step of the urea cycle is the reaction of ornithine with carbamoyl phosphate to produce citrulline. The reaction is catalyzed by ornithine transcarbamoylase.

(a) Write the equation for this transformation, including structures of all reactants.

(b) Write an oranic chemist's curly arrow" mechanism for the reaction as it might occur in vitro.

(c) Describe the mechanism of the enzymatic reaction, including the structure of the enzyme, the catalytic residues, and any intermediates that may be formed. Consult J. Biol. Chem., 1998, 273, 34247 for assistance.

7. The penultimate step in the synthesis of the vitamin thiamin by bacteria involves the reaction of ccarboxythiazole phosphate with hydroxymethyl pyprimidine pyrophosphate catalyzed by thiamin phosphate synthase. Provide structures for the enzyme and the reactants, and write the mechanism by which this reaction occurs. Identify any eukaryotic analogs of the enzyme and provide a comparison of sequences. for assistance: Biochem., 1999, 38, 6460; 2001, 40, 10103; Ann. Rev. Biochem., 2009, 78, 569.

8. In class we briefly discussed the bacterial enzyme subtilisin, and compared its structure (PDB 2st1) with that of the mammalian serine proteases. We suggested that it represented an example of convergent evolution.

Use the sequence to search the NCBI protein sequence database and identify 3-4 other proteins with significant sequence similarity. Provide an alignment of these sequences, and make structural comparisons where possible. Are any of them serine proteases? What can you say about the evolutionary relationship between these proteins?