Mutagenesis & Enzyme Catalysis via Transition State
Part IV. Transition-State in Enzymes & Biotechnology
Another and perhaps the most convincing proof of the existence of the transition-state intermediate in the enzyme catalyzed reaction, was devised by a Professor in England, Allan Fersht, who used biotechnology to approach this question. His method involved site-directed mutagenesis. First, his research group identified the key amino acids at the active site of an enzyme for which they not only had the 3-D structure but also a clone. The cloned gene was expressed in a bacterium and so they could carry on site-directed mutagenesis on the specific AA side chains at the active site and determine what the impact of changing these AA's has on the catalytic properties of an enzyme by study both the natural or wild-type enzyme in comparison with the mutant forms of the enzyme.
The enzyme they were studying is called Tyrosine tRNA synthetase. Tyr tRNA synthetase (or Tyr RS for short) is involved in protein synthesis where the amino acid are attached to the tRNA prior to binding to the ribosome where the tRNA binds to the mRNA and the amino acid is joined to the growing chain of the polypeptide which is being made on the ribosome. Tyr RS catalyzes the following reaction in two steps:
Figure 6. Reactions catalyzed by Tyrosine tRNA Synthetase during the coupling of Tyr to its specific tRNA.
The first of these reactions was selected by Fersht for particular study and it is called the activation step where Tyr's carboxyl group is activated to be reactive using ATP to form a mixed anhydride. For this step, Fersht hypothesized that the reaction would have the following transition-state intermediate:
Figure 7. The reaction where Tyr is activated by ATP in the first step catalyzed by Try RS and the possible transition-state intermediate in the reaction.
Fersht imagined that enzyme must have specific AA side chains in the enzyme's active site for stabilizing this transition-state intermediate and he set out to determine which amino acids side chains in the active site these were and to do site-directed mutagenesis to make mutants of the Tyr RS with different AAs in their places in the active site. This allowed him to develop a very elegant proof of the concept that an enzyme must have a high affinity for its transition-state intermediate.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu