Mutagenesis & Enzyme Catalysis via Transition State
Part II. Transition-State Theory of Enzyme Catalysis
I want to return to a question I have ask before in this class: How do enzymes catalyze reactions? We have addressed that in several ways but we have the same fundamental answer to start from - Like all catalysts, enzymes decrease the energy required to a reaction started. But this answer does not tell us at the molecular level how the enzyme does it and it is at this level that we have worked at finding the answer, which was partly achieved by understanding that enzymes can store energy from the binding of the substrates and use it later to make catalysis more efficient. That is part of the reason why we think enzymes have large structures. We also addressed the question by looking at the enzyme mechanism and saw how AA side chains assist with bond rearrangements. Now we want to apply another theory of chemistry to enzyme catalysis. This theory is the one called transition-state theory. Starting with the transition-state theory of chemical reactions, Linus Pauling suggested that enzyme catalysis can also work by this mechanism (he made this suggestion about 1940 or so). In this concept, an intermediate form exists between the substrates and products in an enzyme catalyzed reaction and this intermediate is called the transition-state intermediate, just as it is in chemistry.
Figure 2. Diagram illustrating the concept of the transition-state intermediate in a chemical reaction.
In the above diagram, A + B are substrates which must get over the energy hump in the reaction pathway which is measured by the 'G' on the y-axis that is called "free energy". On the way to forming products P + Q, the reaction goes through an intermediate which is neither the substrates nor the products and is 'intermediate' between them - so it is called an intermediate and since it is going through the transition from substrates to products it is called the transition-state intermediate. For an enzyme and in enzyme mechanisms, this suggests a new step in the process of catalysis:
Figure 3. The standard enzyme mechanism with the addition of the transition-state intermediate in the process.
So after the ES complex forms and before product P is made, S becomes the transition-state intermediate, which we will call S*Éso ES* must form before EP does. For example, the transition state in the trypsin catalyzed hydrolysis of a peptide bond will be between the key serine's side chain hydroxyl and the carbonyl group of the peptide bond being hydrolyzed. We have also called this the tetrahedral intermediate since the carbon has a tetrahedral shape in this intermediate:
Figure 4. The transition-state intermediate in the trypsin catalyzed hydrolysis of a peptide bond.
This leads to the idea that the enzyme will have a higher affinity for the transition-state intermediate than the substrate and will bind the transition-state intermediate more tightly than substrate. For catalysis to be efficient the enzyme must bind the transition-state more tightly than the substrate or it will simply be a substrate binding protein and not a catalysts. That's why the Ks for the substrate binding to the enzyme is not the same as the Km for the substrate since the Km involves the process of making product, which must go through the transition-state intermediate, as well as the binding of the substrate.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu