Mutagenesis & Enzyme Catalysis via Transition State
Part VI. Transition-State Intermediate Binding in an Enzyme
Well now let us look at the results for changing the side chains of amino acids thought to be involved in stabilizing the transition-state intermediate. First let's review the hypothesis:
Figure 12. Hypothesis for the formation of the transition-state intermediate and the roles of Thr40 and His45 in this process.
The 2 amino acid side chains can form hydrogen/ionic bonds with the pyrophosphate group of the transition-state intermediate and hold them in position during this phase of the catalytic mechanism.
Figure 13. A more detailed model of the role of Thr40 and His45 in stabilizing the transition-state intermediate.
To test if these 2 amino acid side chains have a special role in catalysis via transition-state intermediate stabilization, Thr40 was changed to Ala by site directed mutagenesis and His45 was changed to Gly. The idea is that neither of these changes in side chain would disturb the overall structure of Tyr tRNA synthetase nor change the position of any other groups in the active. Fersht proved this by preparing crystals of these mutants and showing that they had the same 3-D structure as the natural or wild-type enzyme. Then he studied the kinetics of these mutants.
Figure 14. Kinetics of the AA Residues involved in Transition-State Stabilization.
From the results shown in Fig. 14, it is clear that the Thr40Ala and His45Gly mutants were much poorer catalysts than the wild type with the Thr40Ala mutant having a 7000-fold lower catalytic rate constant than the wild-type. When the double mutant was made with both Thr40Ala and His45Gly, the catalytic rate constant was 32,000-fold less than wild-type. In addition, there was little impact of these mutations on the Ks binding constants for the substrates Tyr and ATP; in fact, Tyr was bound more tightly to the enzyme in the mutants than the wild-type, which suggests that taking away the transition-state intermediate stabilizing groups (ie Thr40 and His45) actually does lead to an enzyme form which binds substrate more tightly and is less catalytically active.
In summary, these experiments have shown a way to very precisely and elegantly prove that the stabilization of the transition-state intermediate in an enzyme is critically important to its functionality.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu