Phosphoenolpyruvate carboxykinase (PEPCK) is a family of enzymes with three distinct classes. Two of these classes—ATP-dependent and GTP-dependent PEPCKs—are well-characterized and structurally similar (~70 kDa). The third class, PPi-dependent PEPCK, is relatively understudied despite being both functionally and structurally divergent (~130 kDa). We focus on this divergent class using enzyme kinetics, various biophysical tools, small-angle X-ray scattering, X-ray crystallography, and cryoEM.

Our past and current investigations have revealed that the additional 60 kDa mass results in various interfaces that allow PPi-dependent PEPCK to oligomerize with itself or other paralogs, forming monomers, dimers, tetramers, and filaments (Figure). Each complex is induced through one of three allosteric sites and each oligomeric state displays varying levels of activity, indicating a complex relationship between structure and activity.

Our future goals include:

1. Uncovering the "wiring" of known and unknown allosteric mechanisms that regulate structure and function.

2. Studying new forms of the enzyme with unique sequences or interesting features.

3. Identifying small molecule inhibitors for selectively targeting pathogens with this class of PEPCK.

4. Understanding how structural differences contribute to kinetic variations across the three PEPCK classes.

A long-term goal is to map the evolutionary path of this enzyme family and explore the potential to convert one class into another.

An enzyme's activity and its ability to bind ligands or substrates varies with temperature.  However, the mechanistic origins of enzyme temperature dependence are not well understood.  This is largely due to a lack of multi-temperature structural data, which would allow the changes in enzyme dynamics to be directly observed as temperature is varied.

We have used multi-temperature X-ray crystallography and enzyme kinetics to assign structural states to different temperature regimes. Our data indicate that:

1. Both the enzyme and ligand/substrate change positions and occupancies with temperature. At higher temperatures, the enzyme-substrate complexes generally adopt more catalytically "ready" states.

2. Ligand binding increases with temperature as a result of changes in the active site configuration.

Moving forward, we aim to explore additional systems using a similar approach to not only understand what is changing, but how.  This includes dynamic enzymes whose study will lead to a deeper biophysical understanding of temperature dependence, and those with potential in applied settings like plastic degradation.

Biology thrives across a broad temperature range, from -20°C to 121°C. Since temperature strongly influences both structure and function, thermal pressures will drive adaptation to maintain activity at both high or low temperatures.

We investigate thermally evolved enzyme variants to understand how structural modifications translate into observed kinetic changes. Our studies on psychro-, meso-, and thermophilic GTP-dependent PEPCKs have shown sequence substitutions to a lid domain that must close over the active site to initiate turnover.  The resulting dynamic changes imparted by these substitutions were predicted to create a more temperature-sensitive lid for the psychrophilic PEPCK.

Now, we seek to uncover the general strategies Nature employs to preserve and optimize function across diverse temperature ranges by expanding our investigations to include PEPCKs from all classes as well as other enzyme families.

Structural biology has an ever-expanding toolkit for studying the function, structure, and dynamics of proteins. One tool Matt helped develop in the Thorne Lab (Cornell) is a novel time-resolved X-ray crystallography cryoplunger (Figure), designed to enable high-throughput, time-resolved sample preparation on the benchtop.

This approach uses a linear motor to plunge a crystal through a film of substrate. Upon interaction between the crystal and substrate, diffusion and reaction initiation occur. The speed of the plunge and the height of the substrate above a liquid nitrogen reservoir define a time delay.  Once the sample enters the liquid nitrogen, the reaction is quenched. These samples are stored at cryogenic temperatures, and data is collected at a synchrotron. By preparing samples at various time points, we can capture snapshots of the enzyme in action.

Another tool we use is high-pressure X-ray crystallography. In this method, crystals are pressurized in a cell with diamond windows and while pressurized, data is collected. Pressure perturbation studies can reveal flexible or dynamic regions of the protein, offering valuable insights into how proteins function.

Our philosophy is simple: when a crystal system is amenable, apply the entire toolkit. A comprehensive understanding of enzyme temperature-, time-, and pressure-dependencies can allow researchers to construct a more precise landscape of enzyme behavior, advancing both the understanding of specific systems and the broader biophysics of macromolecules.