VIEWS: ENZYMES    Gale Rhodes
Chemistry Department       University of Southern Maine
Links To Files Used In Biochemistry Class (CHY 361-363)
Topic: Enzymes
For graphics from other topics, see Topics List.
Set your browser to use RasMol for PDB files and RasMol scripts: See Configuring Netscape.
Molecules to Study Lysozyme
Click here for human lysozyme: 1lz1.pdb
Click here for human lyzozyme with bound hexa-NAG: 1lzsA.pdb
NOTES
· Lysozyme cleaves its substrate between the fourth and fifth NAG residues in the hexasaccharide (between NAG139 and NAG140 in 1lzsA.pdb). The structure of 1lzsA.pdb was done at low temperature and pH, and the investigators expected to find intact hexa-NAG at the active site. Instead, they found tetra- and di-NAG, indicating that the enzyme slowly cleaved the substrate, even under non-optimal conditions. The investigators believe that their experiment trapped the enzyme and substrate somewhere on the reaction path between the transition state and product geometry.
· The catalytic residues are glu35 and asp53 (as opposed to asp52 in hen-egg-white lysozyme). Look at the position of these sidechains with respect to the cleavage site.
· If you are using SwissPdbViewer, superimpose the two structures and compare them in the substrate-binding region. This will show you how the conformation of lysozyme changes upon binding its substrate.
Prepared View of Lysozyme Active Site (RasMol Script)
First, click here to download the pdb file of the active site region and hexa-NAG: 1lzsChABndg.pdb
Next click here to download a script showing the details of NAG binding: 1lzsChABndg.spt
   To run this script, you must have the pdb file 1lzsChABndg.pdb, the script file 1lzsChABndg.spt, and RasMol all in the same directory. Run RasMol and type script 1lzsChABndg.spt on the command line. Press <return>. If you are using a Macintosh, you can run a desktop copy of RasMol before downloading the files, and the script should run automatically upon downloading.
· The carbons of catalytic side chains glu35 and asp53 are shown in green.
· Explore the contacts between the NAG units and the enzyme by selecting NAG (select NAG <return>) and then using the dots command to make a transparent space-filling model of the NAG units.
· Select all atoms (select <return>) and choose Wireframe from the Display menu. Then display NAG in Sticks, set picking monitors and look for hydrogen bonds between the enzyme and the NAG units. How many can you find? Using SwissPdbViewer and the file 1lzsA.pdb (above), it took me about 5 seconds to find 13 potential H-bonds to NAG units.
· Measure the distances from the active site carboxyls (carbons shown in green) to the NAG atoms with which they are proposed to interact.
· All of these tasks are much easier to do if you can view in stereo. Click here to learn how.











Chymotrypsin   Click here for gamma chymotrypsin: 2gch.pdb
Click here for gamma chymtrypsin with a tripeptide (chain c) bound at the active site: 8gch.pdb
NOTES
· Gamma chymotrypsin is alpha chymotrypsin with its autolysis products still bound (See Lehninger, Nelson, and Cox, Principles of Biochemistry, 2nd edition, page 235).
· Examine the area around the tryptophan in chain c (8gch.pdb). Can you see why chymotrypsin cleaves the peptide bond on the c-terminal side of aromatic residues?
· If you are using SwissPdbViewer, load the two structures in succession, without moving the first structure before loading the second. They should be superimposed (if not, use Magic Fit). Look for the catalytic triad (his57, asp102, ser195)) in the neighborhood of chain c in 8gch.pdb.
· Calculate the RMS distances of 8gch from 2gch, and color 8gch by RMS. Are there any substantial conformational differences between the enzyme with and without bound tripeptide?
· All of these tasks are much easier to do if you can view in stereo. Click here to learn how.
Prepared Views of Chymotrypsin Active Site (RasMol Scripts)
First, click here to download a PDB file of the tripeptide bound at the active site: 8gchBndg.pdb.
Next, click here to download a script showing the catalytic residues and the binding pocket: 8gch-01.spt.
Finally, click here to download another view of the active site: 8gch-02.spt.
   To run these scripts, you must have the pdb file 8gchBndg.pdb, the script files, RasMol all in the same directory. Run RasMol and type script 8gch-01.spt or script 8gch-02.spt on the command line. Press <return>. If you are using a Macintosh, you can run a desktop copy of RasMol before downloading the files, and the scripts should run automatically upon downloading.
NOTES:
· In both views, carbons in the side chains of the catalytic "triad", ser195, his57, and asp102, are shown in green. Carbons of the bound tripeptide (designated chain c in the pdb file) are shown in pink. In the first script, measure the distances between the side-chain atoms that are closest to each other in the catalytic triad.
· One of the carboxylate oxygens of the bound tripeptide is not shown, and the carboxyl carbon is very close to the -OH of serine 195. The arrangement is almost the same as in the proposed acyl-enzyme intermediate of the hydrolysis reaction.
· The second script shows the tripeptide in stick form, with surrounding atoms space-filling. Note how the tryptophan of the tripeptide fits into the hydrophobic pocket adjacent to the catalytic site. Display chain c in space-filling form, and slab through the structure to see how the tryptophan side chain fits within the specificity pocket. Trp, phe, and tyr side chains bind best in this pocket, and as a result, chymotrypsin cleaves protein chains on the C-terminal side of trp, phe, and tyr.
· In trypsin, this pocket has a negatively charged aspartic acid side chain in the bottom of the pocket. Postively charged side chains, like those of lys and arg, bind best in this pocket. As a result, trypsin cleaves protein chains on the C-terminal side of lys and arg.
· In elastase, this pocket is much smaller. Elastase cleaves next to small residues like gly and ala.
Tyrosyl-tRNA Synthetase Click here for the enzyme bound to tyrosine: 3ts1.pdb
Click here for the enzyme bound to tyrosyl adenylate: 4ts1.pdb
NOTES
· For a brief discussion of this enzyme, see Lehninger, Nelson, and Cox, Principles of Biochemistry, 2nd edition, page 227ff.
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