How do enzymes “work”? How did they get to be this way? How will they adapt in the future?

Enzymes are the primary functional molecules in cells, providing enormous rate enhancements, specificity and regulation to the diverse chemical reactions that are necessary for life. Disruption of enzyme function, whether through mutation or dysregulation, often results in disease. Enzymes, like all biological components, have evolved to operate within the intricate cellular environment and specific ecological niches. Therefore, a mechanistic understanding of enzyme function and evolution is crucial to understanding biology. Moreover, enzymes hold immense potential in medicine, serving as targets for anti-cancer, antimicrobial, and antiviral drugs, as well as therapeutic agents for metabolic disorders. They also find applications in industry, such as the production of important chemicals and as catalysts for bioremediation. Our central premise is that a quantitative, mechanistic understanding of enzyme function and evolution is critically needed to deeply understand biology and to precisely manipulate enzymes.

 However, achieving a broad mechanistic understanding of enzyme function and evolution necessitates the development of new tools. Enzymes are highly interconnected, with multifaceted functions, and operate in complex cellular environments. Traditional biochemistry, while powerful, falls short when it comes to identifying the numerous residues that contribute to enzyme function beyond the active site, requiring investigations on a scale surpassing traditional methods. In the Pinney Lab, we employ quantitative, high-throughput assays to explore enzyme function, achieving unprecedented breadth and depth. Our lab focuses on the following areas:

  • Biochemistry in the Genomic Age: Expanding the Toolkit for Quantitative High-Throughput Biochemical Assays

    We use microfluidic devices containing >1,500 valved reaction chambers aligned to printed plasmid arrays. These devices allow us to express, purify and assay designed libraries of thousands of enzyme variants all on a single microfluidic device. These devices are unmatched in their ability to parallelize and repeat assays with different substrates and conditions to efficiently generate high-dimensional biochemical data. We are currently expanding the capabilities of these devices and are using them to dissect enzyme function and evolution.

  • Identifying the "Rules" of Enzyme Function and Evolution.

    Understanding how enzymes evolve and adapt can teach us the fundamental principles of how enzymes work. We use a combination of bioinformatic methods to identify putative adaptive changes in enzyme sequences and high-throughput biochemistry to quantify how these sequence changes influence enzyme biochemistry (e.g., kcat, KM, and Ki) at an unprecedented scale.

    Our primary focus is on enzyme temperature adaptation for two reasons. First, temperature influences many important properties of enzymes, such as their activities, stabilities and dynamics. Thus, enzyme temperature adaptation provides a window into the mechanisms governing these properties. Second, understanding enzyme temperature adaptation can give us insights into how organisms will adapt to changing climates at the molecular level.

  • Dissecting Enzyme Function at the Systems & Organism Levels

    Enzymes evolved to operate within the complex environment of an organism, where they form higher-order complexes, are involved in metabolic networks, and are regulated at multiple levels. As these features are not captured by in vitro experiments, we are developing methods to map between enzyme function and organism fitness in high-throughput to understand the biochemical determinants of fitness and parameters critical for pathway optimization.

  • Understanding the Biochemical Mechanisms of Disease Processes

    Most disease variants in enzymes are uncharacterized at the biochemical and cellular levels. With our high-throughput biochemical methods, we can biochemically characterize thousands of disease variants of an enzyme and then map these biochemical effects to cellular fitness, information key to understanding the molecular underpinnings of disease and to designing personalized treatments.