MacDNAsis of Pyruvate Kinase

(This web page was produced as an assignment for an undergraduate course at Davidson College.)

The cDNA encoding for pyruvate kinase was analyzed using MacDNAsis, and the following information was obtained.

Largest Open Reading Frame

               Figure 1: a series of 3 open reading frames for Mus musculus pyruvate
               kinase, as determined by MacDNAsis. The stack has frame 1 on top and
                 frame 3 on bottom. The red triangles indicate start codons, green lines
                 indicate stop codons, and the white boxes are open reading frames. The
                 largest open reading frame is indicated in blue.

You can find the DNA sequences that code for pyruvate kinase for five organisms by clicking here.

When translated into its predicted amino acid sequence, Mus musculus pyruvate kinase has a predicted molecular weight of 58.01 kDa.  See the predicted amino acid content of the enzyme by clicking here.


Hydropathy Plot

                Figure 2:  A Kyte-Doolittle hydropathy plot of the predicted
                Mus musculus pyruvate kinase. Hydrophobic portions are above the
                horizontal line; hydrophilic portions are below the line.

For Kyte-Doolittle plots, possible membrane-spanning domains are indicated by values above 1.8.  This enzyme has many such instances, including (approximately) those close to residues 10, 60, 105, 240, 410, 440, 460, 480, and 515.  Apparently, pyruvate kinase is quite membrane-associated, as it has multiple membrane-spanning domains.

To learn a little more about Kyte & Doolittle, see this page by Biotools Incorporated.

Antigenicity Plot

                Figure 3:  A Hopp-Woods antigenicity plot of the predicted Mus musculus
                pyruvate kinase.  Hydrophilic portions are above the horizontal line;
                hydrophobic areas are below the line.

For Hopp-Woods plots, possible antigenic areas are those that are most positive, indicating the greatest degree of hyrophilicity.  These areas are the epitopes most likely to bind to antibodies.  This graph can help determine which portion of the enzyme to use in order to generate a peptide for synthesis of a monoclonal antibody against the peptide.  This antibody will recognize a linear epitope while the enzyme is in its native conformation.  The best possible candidates are areas near residues 160, 180, and 230, although many other areas (indicated by positive spikes in the graph) may possibly be antigenic.  For this enzyme, I would generate a peptide from residues 175-195, because that spike seems to be the most hydrophilic.  Another option would be to use residues 340-360 because --although not as high as the spike near residue 180-- that is the broadest hydrophilic region of the protein.

For a summary on Hopp & Woods, check out this page from Biotools Incorporated.


Predicted Secondary Structure

The predicted secondary structure of mouse pyruvate kinase is given below.  It is derived from the amino acid sequence generated previously (click here to see the amino acid sequence in a text format).

                    Figure 4:  The predicted secondary structure of Mus musculus
                    pyruvate kinase.  The structure legend is given directly
                    above the figure. The structure consists of two turns and
                    multiple helixes, sheets, and coils.

Sequence Similarity

The sequences of five organisms were analyzed and compared to find similarities.  Of the five organisms, three were very similar in sequence, while the other two were less similar.  The organisms analyzed were Saccharomyces cerevisiae, Oryctolagus cuniculus, Mus musculus, Homo sapiens, and Chlamydia trachomatis.  Click on the species to see their amino acid sequences in a text format.

            Figure 5:  A comparison of partial pyruvate kinase sequences using
            the one-letter amino acid code, from top to bottom:  protozoa, human,
            mouse, rabbit, and yeast.  Aligning residues appear as yellow text
            against a black background, non-aligning residues are blue against a
            white background.

It is apparent from the figure that the highest degree of similarity is among the mammalian pyruvate kinase amino acid sequences.  The protozoan and yeast protein sequences are not very similar in sequence.



In order to examine the degree of amino acid conservation over time, a phylogenetic tree was constructed using all 5 proteins, from human, mouse, rabbit, yeast, and protozoa.  (click to see the amino acid sequences)

Figure 6:  A phylogenetic tree showing the genetic distance between each of the sequences, from top to bottom:  human, mouse, rabbit, yeast, and protozoa.

From this data, it is apparent that the genetic distance between mammals is very short; they diverged very recently along the evolutionary timeline.  On the other hand, yeast and protozoa seem to have diverged much longer ago.  These results are not surprising, as they are very consistent with modern theories of evolution.

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