Volume 4, Issue 31 September 2007
Directing Enzyme Evolution
Professor Jack Kirsch is also affiliated with the California Institute for Quantitative Biosciences (QB3). Photo courtesy of Jack Kirsch
Enzymes are a picky lot. Of the many thousands of molecules drifting through their environment, most enzymes will react with only one-its preferred substrate, or target.
The secret to that specificity is buried in the nooks and crannies of an enzyme's active site. The amino acids lining this pocket both help an enzyme bind to its substrate and catalyze a chemical reaction. The positions of these amino acids, in turn, are controlled by genes.
"We want to figure out how to discover the few most important gene mutations that will change enzyme specificity," says Jack Kirsch, Professor of the Graduate School Division of Biochemistry and Molecular Biology. "But we don't know which changes will give it the new activity."
A Venn diagram comparing the amino acid positions common to many species' versions of the related enzymes AATase (left) and TATase (right).
Understanding how to design enzymes to work on desired targets would be a great boon to industry and basic science alike. Once scientists solve this problem, bespoke enzymes for the design of more effective drugs, enhanced detergents, and even the creation of biofuels won't be far behind.
Kirsch studies enzyme specificity using a process called directed evolution. He has developed a way to combine logic and natural selection to custom design enzymes that will react with new substrates. Kirsch says, "You take an enzyme that has a particular function and try to get it to evolve under laboratory conditions so that it acquires the function of another."
In one recent project, Kirsch and his laboratory set out to morph an enzyme involved in the metabolism of one amino acid into a related enzyme that reacts with a different amino acid.
To analyze which mutations were most important to make, Kirsch borrowed an idea from his son's eighth-grade math classes. Using the Venn diagrams of set theory, Kirsch compared the sequences for both enzymes in creatures from worms to humans. He found that certain amino acids were more likely to occupy the same position in both enzymes.
A native of Detroit, Kirsch explains the concept in terms of cars. He says, "Suppose you wanted to convert a Volkswagen into a Cadillac. Certain things are going to be the same; both cars have a motor, four wheels, a chassis, and a steering wheel. The big difference is in the passenger compartment. The Volkswagen's is rather small and uncomfortable; the Cadillac's is big and roomy. This is where you're going to start remodeling."
The evolutionary path of enzymes can affect how easy it is to alter their specificity. This diagram illustrates the evolution of two enzymes, AATase and TATase.
Because Kirsch's mutant enzyme and its natural counterpart had comparable behavior, logic suggests they should be nearly identical. In fact, most of the changed amino acids in the mutant were different from those in the natural version.
The discrepancy, Kirsch says, can be chalked up to evolution. The natural enzyme and Kirsch's mutant evolved from very different starting materials. The first enzyme gave rise to the second some 1 billion years ago. By the time Kirsch and his lab began their experiments, the modern enzyme he was working with looked very different from that early ancestor. It was no longer possible to recreate each step of its original evolutionary path.
A previous researcher, John Holbrook of the University of Bristol, encountered the same conundrum in the 1990s. Though he had managed to make one enzyme act like its relative with a single mutation, Holbrook found it impossible to reverse the experiment.
Kirsch's laboratory has since revisited the Holbrook problem. Armed with current enzyme evolution data, and using Venn diagrams to identify the most critical parts to remodel, they used their directed evolution approach to produce a good target enzyme with just five amino acid replacements.
Kirsch's techniques bring scientists one step closer to being able to aim these molecular missiles at will. Kirsch adds, "Understanding how to identify these specificity determinants is important not only for understanding protein evolution, but for learning how life evolved and continues to evolve."