Tracing enzyme mechanisms across evolution to elucidate the origins of enzymatic catalysis

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Many biologically important reactions occur spontaneously at rates that are far too slow to sustain life. Enzymes are the naturally occurring catalysts that are responsible for accelerating these reactions into the realm of biological significance. The accelerations provided are often profound, with enzyme-catalyzed reactions occurring up to 1023-fold faster than the corresponding spontaneous processes. In one case, a reaction that would otherwise require 80 million years is completed by an enzyme in 18 milliseconds. While the magnitude of this catalysis has attracted significant attention, an understanding of how enzymes catalyze reactions so efficiently has remained elusive. This program will elucidate the specific determinants of enzymatic catalysis by extending the question of “how do enzymes work?” to encompass a larger, more interesting one: how do enzymes evolve to work so efficiently? By characterizing enzyme mechanisms over the course of evolution, our program will provide the first unambiguous demonstrations of the specific changes that correlate with gradually increasing catalytic efficiency. Whereas traditional enzymology makes inferences about the origins of enzymatic catalysis from studies of singular, modern enzymes, our program will introduce the dimension of evolutionary time to the study of enzyme mechanisms to evaluate the features that are important to catalysis. This program will provide significantly more detail about the origins of enzymatic catalysis, just as a movie provides more detail than a still image. This proposal will use a combination of kinetic isotope effects and computation to evaluate how enzymes evolve both naturally and in the laboratory. Evolutionary lineages of natural, extant enzymes will be determined using ancestral sequence reconstruction. This technique will effectively permit the resurrection of enzymes that have been “dead” for billions of years and subsequent biochemical characterization of these species along their natural lineages. In the lab, enzymes can be forced to evolve into more efficient catalysts. This “directed evolution” is a powerful technique, as shown by the 2018 Nobel Prize in Chemistry, but it remains a "black box". Our program will demonstrate the precise mechanisms by which directed evolution leads to more efficient enzymes and will lead to improved design principles for novel biocatalysts. This program will use an innovative approach to address the fundamental question of how enzymes work. We cannot expect to efficiently engineer what we do not understand, and by better understanding how enzymes work, we will facilitate efforts to produce de novo biocatalysts with useful activities that will benefit the Canadian chemical industry. This work will also strengthen Canada's reputation as a leader in the fields of physical organic chemistry and enzymology and will produce HQP who will be well prepared to contribute to the Canadian scientific community in academia and industry.