Examples:

Human Lysozyme

• Lysozyme cleaves its substrate between the fourth and fifth NAG residues in the hexasaccharide (between NAG139 and NAG140 in 1LZS). The structure of 1LZS 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 asymetric unit of this model contains two lysozyme molecules, the second containing only tetra-NAG).
• 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.
• Chain A of 1LZS is superimposed on 1JSF by Magic Fit followed by Improve Fit. Compare 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.
• Explore the contacts between the NAG units and the enzyme by selecting the NAG and then displaying only the neighbors within 5 angstroms.
• Use Tools: Compute H-bonds to find the hydrogen bonds between the NAG units and the enzyme.
• 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.

Chicken Lysozyme

• On the human model (1JSF), residues are colored according to comparison with corresponding residues on the chicken model (1HEL). CPK-colored residues are the same in human and chicken; green represents conservative substitutions, and purple represents nonconservative substitutions (as defined by SPV). (How would you achieve this color scheme with SPV? Some hints in the last question of this section.)
• When you open this file, only the nonidentical residues are selected. With 1JSF active, click on the surface heading of the control panel (the little square of dots with a small "v" under it. This adds van der Waals surface to residues currently selected, thus making the substitutions stand out. Notice that differences are uniformly distributed over the model, except that the active site cleft looks somewhat empty. Zoom in on th active site; you will find many CPK-colored residues -- residues that are identical in human and chicken lysozyme. Is this what you would expect?
• Make 1JSF the active layer and use Select: aa Identical to ref... to identify the residues that are identical in the two models. The Layer Infos window shows you how many residues are selected. Use Select: aa Similar to ref... . Now the number of selected residues is the number of identical and conservatively substituted residues between the two models.
• What specific substitutions do you see in the residues in the active-site cleft? Are the substitutions conservative or nonconservative?
• Can you find an easy way to color 1JSF as shown? Hint: Useful commands are Select: Inverse Selection and the other Select commands mentioned above. Try to use these commands to color 1HEL just like 1JSF is colored.

Chymotrypsin

• Examine the 4CHA layer. The residues of the "catalytic triad" are labeled. Compute H-bonds to see how they interact. Other residues shown in wireframe make up the pocket that determines the specificity of chymotrypsin.
• In the 6CHA layer, examine the area around the PBA inhibitor (has surface dots). Can you see why chymotrypsin cleaves the peptide bond next to bulky aromatic residues?
• Calculate the RMS distances of 6CHA from 4CHA, and color 6CHA 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.

Trypsin

• In 2PTN, note the arrangement of the catalytic groups. Without a ligand shown, can you identify the binding region for the ARG or LYS side chain for which trypsin is specific? Blink to 1TPA to see if you are right. A lysine of BPTI fills the pocket in 1TPA. BPTI prevents trypsin from becoming active before it is secreted into the small intestine. It binds to trypsin much like a substrate, but causes conformational changes that prevent trypsin from cleaving it.
• Identify the scissile peptide bond in BPTI (or what would be the scissile bond... .)
• Turn off ribbons to see the catalytic groups and a few residues of the inhibitor. Select the visible groups and add their neighbors within 6 angstroms. Be sure to turn on the side chains for visible groups. What specific interactions make the lysine sidechain welcome in the specificity pocket?
• Note especially the distance from the BPTI lysine-NH2 and the carboxyl of ASP189. This is not an ideal distance for a salt bridge. Why might this interaction be non-ideal?
• Color 1TPA by RMS differences between the BPTI complex and the apoenzyme. This will make BPTI red, because it is absent from the other layer. Is there evidence of conformational change when BPTI binds? Can you see any reasons why this complex does not cleave BPTI?

Mammalian Serine Protease Family Portrait

• SPV superimposed the models by Magic Fit. Blink through the models to compare their overall structure.
• Zoom in on catalytic triads and compare them.
• By now, you should be able to identify the specificity pockets of the three enzymes. Display residues there, and compare the interiors of the pockets.
• Look up the term oxyanion hole in your textbook. Can you find the residues that form the oxyanion hole in these models? Phospholipases have the same task of stabilizing tetrahedral transition states during ester hydrolysis.

Zymogen Activation: Chymotrypsinogen to Chymotrypsin

To simplify the view, use Prefs: Ribbons to reduce the ribbon display to a single strand. You can also turn off labels by shift-clicking any checkmark in the label column of the Control Panel, and if you do not alter the selections, you turn them back on again by clicking the heading of the label column. Compute H-bonds in all layers. Blink through the models. In succession, you see chymotrypsinogen (the zymogen), chymotrypsin (the active enzyme); and enzyme+PBA, with the phenyl ring of PBA occupying the specificity pocket. Use this blinking cycle to explore how activation affects the catalytic triad, the shape of the specificity pocket, the positions of side chains that line the pocket, and the positions of residues that are affected by the cleavage of the zymogen when it is activated.

NOTES

• Does activation of the zymogen change the position or hydrogen bonding of any groups in the catalytic triad?
• Does activation affect the shape of the backbone in the specificity pocket?
• Does activation affect the positions of sidechains that line the pocket?
• When the zymogen is activated, a new amino terminus is produced at ILE16. What happens to this new terminus and to ASP 194?
• Cleavage also occurs at TYR146. What happens to the side chain of its neighbor, ARG145, upon activation?
• Display 2CGA and 6CHA simultaneously, but display only PBA1 of 6CHA. Does PBA fit the specificity pocket of the zymogen as well as it fits the active enzyme?
• Color backbone and sidechains in all three layers by B-factor. In well-refined models, B-factors are lowest (blue) where a model is well-ordered, and higher (green, yellow, red) where the model is less ordered. Can you discern whether the specificity pocket is better organized in the zymogen or the active enzyme?

Subtilisn

• Blink to the 4CHA layera and zoom in on the catalytic triad. Blink between the models. What differences can you find between the arrangements of the catalytic residues?
• Look at the ribbon diagrams as you blink them. Are chymotrypsin and subtilism similar in overall conformation?
• What does is mean to say that chymotrypsin and subtilisn are examples of convergent evolution?

Aspartate Transcarbamoylase

• Blink between the T-state and R-state layers to see the differences between the two states.
• What colors are the regulatory and catalytic subunits?
• The T-state model includes an allosteric effector. Identify it, and note its location.
• Is the allosteric effector bound to the catalytic or the regulatory subunit?
• The R-state model includes a substrate analogue, which binds at the active site. Note its location.
• Is the substrate analog bound to the catalytic or the regulatory subunit?
• How do the conformational changes between the two states alter enzyme activity?
• Study the details each binding site by selecting one effector or substrate analog in the Control Panel and using Select:Neighbors of Selected aa to display interacting residues.