Mutagenesis & Enzyme Catalysis via Transition State
Part III. Transition-State Analogs as Very Good Enzyme Inhibitors
One way to try to prove that the transition-state intermediate exists in an enzyme catalyzed reaction and is part of the enzyme catalyzed mechanism, is to design inhibitors of an enzyme that look like what you think the transition-state intermediate for the enzyme catalyzed reaction looks like. Furthermore, to prove the idea that the enzyme binds the transition-state intermediate more tightly than it does the substrate, we should compare these transition-state analogs (the transition-state like inhibitor molecule) to competitive inhibitors of the enzyme, which of course look like the substrate chemically, and see which is the stronger inhibitor. An example of this type of experiment is found in the studies of lysozyme:
Figure 5. Lysozyme and its transition-state analog as an inhibitor. In this enzyme, the transition-state intermediate is thought to be a planar carbonium ion and so a transition-state analog which should be a very good inhibitor would be a sugar with a planar carbon at the reactive center, which is found in a lactone form of the sugar. The lactone sugar is a much better inhibitor than a sugar with the 'chair' shaped reactive center like is found in the substrate or competitive inhibitors. In general, it has been found that transition-state analogs are the best inhibitors of enzymes - they often have Ki's 1000 times or more smaller than competitive inhibitors (smaller Ki indicates tighter binding to the enzyme's active site).
Another way to address the issue of the importance of the transition-state intermediate in enzyme catalysis is to design a protein which will strongly bind a molecule which represents the transition-state intermediate in a reaction you wish to make a catalysis for. So if the protein binds the transition-state analog very well, then it should be a catalyst for the reaction having that transition-state intermediate. This has been done by using antibodies to make the catalyst like an enzyme or in other words make a 'catalytic antibody'. Since an animal can be immunized or induced to make an antibody against essentially any chemical, if the animal is immunized with the transition state analog, will it be an enzyme for the reaction with that transition-state? For example, phosphate esters are tetrahedral in shape and could be a transition-state analog for an enzyme that hydrolyzes carbon-based esters. To test this hypothesis, an animal was immunized with a phosphate ester containing protein and then the antibodies were isolated from the animal's serum. These purified antibodies indeed did have esterase activity and so a catalytic antibody can be made by immunization with the transition-state analog.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu