Mutagenesis & Enzyme Catalysis via Transition State
Part V. Site-Directed Mutagenesis of an Enzyme
But first, before Fersht could study the part of the active site involved in stabilizing the transition-state intermediate, he needed to establish which amino acid side chains were involved in binding the substrate and catalyzing bond rearrangements. This is pretty much like what I have already described for the serine proteases and a number of other enzymes in this course. But Fersht went one step further in his studies, he made a series of mutants of these active site AA residues and studied what impact changing key AAs involved in substrate binding and other aspects of catalysis. First, he hypothesized what AA side chains in the active site were doing what:
Figure 8. Hypothesis for the catalytic mechanism of Tyr tRNA Synthetase and the role of key amino acid side chains at the active site.
In this model, 7 amino acid side chains were identified at the active site: 2 Tyrosine residues (Tyr34 and Tyr169) were suggested to be involved in binding the substrate Tyr; Cys35, His48 and Thr51 were suggested to be involved in binding the adenosine part of the ATP substrate; and finally, Thr40 and His45 were expected to be involved with stabilizing the transition-state intermediate. In the catalytic mechanism, the resting enzyme ready to start catalysis is shown in part a of Fig. 8 with the AA side chains list above shown surrounding three pockets in the active site. First, the Tyr binds to its binding site in part b; next ATP binds to its binding site and the reaction starts by Tyr attacking the alpha-phosphate of the ATP as shown in part c. This leads to the transition-state intermediate, which is shown in part d with the terminal two phosphates of ATP held in the special pocket of the active site where Thr40 and His45 are located. Next, the transition-state intermediate breaks down into products as shown in part e. Finally, the products leave the active site, with the pyrophosphate (PP) leaving first followed by the activated Tyr-AMP which is shown in part f and finally going back to part a where the resting enzyme is ready to start the cycle over again.
Figure 9. The 3-D structure of Tyr tRNA Synthetase with the region of the active site identified.
The active site of the enzyme was analyzed by modeling where the product of the reaction was fitted to all the possible hydrogen bonding groups available in the active site. This showed them which amino acids to target for site-directed mutagenesis to determine if altering them to other amino acids would impact the catalytic functionality of the enzyme.
Figure 10. Model of the Active Site of Tyr tRNA Synthetase with the Product, Tyr-AMP, bound.
In this model, I have selected 4 of the residues Fersht's group studied by site-directed mutagenesis and identified the residues which these were changed to. 1. Tyr169 was changed to Phe so that it lost the ability to make a hydrogen bond with the substrate Tyr, but retained its ability to function structurally. 2. Tyr34 was changed to Phe for the same reason as Tyr169. 3. Cys35 to Ser which gives it similar chemistry but the OH on Ser is smaller than the SH on Cys and Cys35 was also changed to Gly which is completely removing the side chain. 4. His48 was changed to Gly also which eliminates its side chain. Then they analyzed the kinetic properties of the mutant forms generated by these changes in comparison to the natural or wild-type enzyme.
Figure 11. Kinetic Properties of Mutants of AA Residues involved in Substrate binding in Tyr RS.
When the 2 Tyr residues in the Tyr substrate binding site were changed to Phe, little impact was found on the k3 or catalytic constant (which you remember is sort of like the Vmax) but Ks for Tyr was increased by 2-fold when in the Tyr34Phe mutant and by over 100-fold Tyr169Phe mutant. For the Cys35 mutants, the impact was mainly on the catalytic rate constant and there was little impact on the Ks for ATP, which was also true for the His48Gly mutant. So with any of these mutants, only a small change was found in kinetic properties of the enzyme but it is sufficient to show that these AA side chains are important in catalysis. The magnitude of these changes in kinetic properties will become important next when we look at mutants in the other active site residues - so let's keep in mind that the biggest change was in the rate constant by about a factor of 10 when Cys35 2as changed to Gly.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu